Download Local Gene Expression in Axons and Nerve Endings: The Glia

Physiol Rev 88: 515–555, 2008;
doi:10.1152/physrev.00051.2006.
Local Gene Expression in Axons and Nerve Endings:
The Glia-Neuron Unit
ANTONIO GIUDITTA, JONG TAI CHUN, MARIA EYMAN, CAROLINA CEFALIELLO, ANNA PAOLA BRUNO,
AND MARIANNA CRISPINO
Department of Biological Sciences, University of Naples “Federico II,” and Stazione Zoologica “Anton Dohrn,”
Naples, Italy
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I. Introduction
II. Origin of Axonal and Presynaptic Proteins: The Cell Body Hypothesis
III. Local Protein Synthesis in Axons
A. Early evidence
B. Model systems
C. Vertebrate axons
D. Other axons
E. Neurons in culture
IV. Local Protein Synthesis in Nerve Terminals
A. Model systems
B. Vertebrate nerve endings
V. Synthesis of Axonal and Presynaptic RNA: The Cell Body Hypothesis
VI. Local Synthesis of Axonal RNA
A. Model systems
B. Vertebrate axons
VII. Local Synthesis of Presynaptic RNA
A. Early evidence
B. Model systems
C. Vertebrate nerve endings
VIII. Transfer of Glial RNA to the Neuron Cell Body: The Glia-Neuron Unit
A. A brief overview
B. Experiments with single cells
IX. The Local System of Gene Expression in Axons and Nerve Terminals
A. Axons
B. Nerve terminals
X. Conclusion and Novel Perspectives
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Giuditta A, Chun JT, Eyman M, Cefaliello C, Bruno AP, Crispino M. Local Gene Expression in Axons and
Nerve Endings: The Glia-Neuron Unit. Physiol Rev 88: 515–555, 2008; doi:10.1152/physrev.00051.2006.—Neurons
have complex and often extensively elongated processes. This unique cell morphology raises the problem of how
remote neuronal territories are replenished with proteins. For a long time, axonal and presynaptic proteins were
thought to be exclusively synthesized in the cell body, which delivered them to peripheral sites by axoplasmic
transport. Despite this early belief, protein has been shown to be synthesized in axons and nerve terminals,
substantially alleviating the trophic burden of the perikaryon. This observation raised the question of the cellular
origin of the peripheral RNAs involved in protein synthesis. The synthesis of these RNAs was initially attributed to
the neuron soma almost by default. However, experimental data and theoretical considerations support the
alternative view that axonal and presynaptic RNAs are also transcribed in the flanking glial cells and transferred to
the axon domain of mature neurons. Altogether, these data suggest that axons and nerve terminals are served by a
distinct gene expression system largely independent of the neuron cell body. Such a local system would allow the
neuron periphery to respond promptly to environmental stimuli. This view has the theoretical merit of extending to
axons and nerve terminals the marginalized concept of a glial supply of RNA (and protein) to the neuron cell body.
Most long-term plastic changes requiring de novo gene expression occur in these domains, notably in presynaptic
endings, despite their intrinsic lack of transcriptional capacity. This review enlightens novel perspectives on the
biology and pathobiology of the neuron by critically reviewing these issues.
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I. INTRODUCTION
FIG. 1. Schematic representation of an adult vertebrate neuron
whose axon corresponds to a length of ⬃1 cm. Several neurons have
axons longer than 1 m and may form thousands of synapses in their
terminal arborization. [Modified from D’Angelo and Peres (64).]
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Neurons relay signals among different regions of the
body that are often distant from each other. Accordingly,
to perform their function, many neurons need to grow
long and branching processes. During the course of phylogenetic evolution, this led to the formation of nerve
cells whose cytoplasm resides largely in the axon domain,
at distances up to meters away from the nucleus. As a
result, the combined mass of cell processes markedly
exceeds that of the cell body in many neuronal types and
many animal species (Fig. 1). Accordingly, neurons account for half the mammalian brain mass, although their
number is only a small fraction of the number of glial
cells. The markedly imbalanced cytoplasmic distribution
between soma and axon domain could not be without
consequence on the trophic support of the axonal periphery. How could a cell body metabolically sustain an axon
comprising more than 100-fold its mass? How could a
perikaryon synthesize all the axonal, presynaptic, and
dendritic proteins in addition to its own? How could it
deliver them to the right place at the right time? These
considerations may not have been in the mind of the
scientists formulating the neuron doctrine (17).
A key problem of cell biology was thus set forth from
the very beginning in terms of two opposing points of
view, two sides of the same coin. One of them centered on
the axon’s trophic dependence on the cell body. The
evidence for such dependence was so compelling that for
a long time it obscured the other horn of the dilemma,
that is, the limited capacity of the neuron soma to satisfy
the trophic needs of the increasing axonal mass (315). As
a result, the cell body was assigned the role of the only
trophic center of the neuron, most people not realizing
that such a view was strongly biased towards one side of
a coin. But the other side did in fact exist, as “not to see
belongs to the observer, while not to exist belongs to the
object” (4). In fact, with time, the initial view started to be
challenged by theoretical considerations and by experimental evidence, and the biological issue moved towards
a more balanced assessment.
This review is an attempt to place in perspective the
historical roots and branches of the main features of the
local gene expression in the axon domain. We will critically describe early and recent evidence, including the
prevailing views and counter criticisms. In our opinion,
this approach will provide a more balanced and realistic
perspective that is likely to lead to a better understanding
of the biology of neurons and glia. The novel concept
prompted by available data suggests that the trophic capacity of the neuron soma may satisfy the needs of its
cytoplasmic mass only up to a certain limit depending on
the ratio of the perikaryal trophic capacity to the cytoplasmic mass and needs. Beyond this limit, which is often
exceeded in many mature neuronal types such as vertebrate sensory and motor neurons, the trophic needs of the
axon domain must be supplemented by periaxonal and
perisynaptic glial cells. Our discussion will be more focused on the latter issue, that is, on the glial synthesis and
delivery of transcripts (as well as proteins and other
cellular components) to the translation system of axonal
sites. Accordingly, data regarding neurons with a limited
axon domain, such as developing or cultured nerve cells,
will be mentioned only within the scope of the review.
Likewise, we will only briefly discuss the growing literature regarding protein synthesis in dendrites (for reviews,
see Refs. 141, 222, 276, 291).
The validity of this view depends on the following
experimental evidence: 1) that protein synthesis occurs in
axons and nerve terminals, 2) that axonal and presynaptic
RNAs are synthesized in periaxonal and perisynaptic glial
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
cells, and 3) that local synthesis of axonal and presynaptic protein is modulated by local transcription processes.
This evidence will be discussed separately for axons and
nerve endings because of the different functional roles of
these two domains and the greater weight of the available
experimental evidence for axons.
II. ORIGIN OF AXONAL AND PRESYNAPTIC
PROTEINS: THE CELL BODY HYPOTHESIS
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structures were often interpreted otherwise, e.g., as glycogen granules or cross-sections of cytoskeletal elements.
It should be added that for quite a time, the lack of
ribosomes was one of the criteria used to discriminate a
central nervous system (CNS) axon from a dendrite. Obviously, such an assumption prevented the identification
of axonal ribosomes. The presumed lack of axonal ribosomes remained for a long time the single most important
reason to object to the existence of axonal protein synthesis. As a corollary, rRNA and mRNA were also assumed
to be absent from the axon domain. Indeed, when axoplasmic 4S RNA was identified by its electrophoretic mobility in
squid (Loligo pealii) and Mixycola (a nematelminth) giant
axons (31, 199), the presence of minor amounts of rRNA was
overlooked. As a result, the widely publicized presence of
only 4S RNA in axons became an additional strong reason
to believe in their incapacity to synthesize proteins.
This established opinion was substantially strengthened by the demonstration that axoplasmic organelles
and matrix were slowly moving distally, as shown by their
massive accumulation on the proximal side of nerve constrictions (325). These key observations stirred a flurry of
experiments aimed at the characterization of what was
known as axoplasmic flow or transport (125). Of the main
features of this complex process, we will only note its
gross distinction in two main rate groups, ranging from a
few millimeters per day (slow axoplasmic transport; Ref.
32) to several hundred millimeters per day (fast axoplasmic transport). Cytoskeletal and cytosolic proteins were
believed to reach the axon by the slow flow, while smooth
vesicles, mitochondria, and other cell organelles targeting
nerve endings traveled with the fast flow (125). While the
mechanism of the fast transport was elucidated in detail
(142, 266), the interpretation of the experimental data for
the slow axoplasmic flow remained controversial (8). In
the experiments using radiolabeled protein precursor, the
slow anterograde flow of radioactivity was initially attributed to the movement of newly synthesized somatic proteins. This interpretation was based on the unproven
assumption that axons lacked the capacity to synthesize
proteins. However, after the demonstration of an axonal
system of protein synthesis (7, 120, 183, 262, 306), these
data could be explained in a radically different scenario,
involving 1) the incorporation of radiolabeled amino acids into the proteins of nerve cell bodies and initial segments of the axon, 2) the release of radiolabeled amino
acids from the degraded axonal proteins, and 3) the distal
diffusion and reincorporation of the radiolabeled amino
acids into the proteins of more distal axon segments. This
explanation was a better fit for the progressive changes in
the distribution of radiolabeled axonal proteins as the
radioactive wave became wider and flatter with time and
kept losing most of its initial radioactivity. For an extensive discussion of this alternative explanation, see Alvarez
et al. (7).
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One of the main experimental bases of the neuron
doctrine (17) regarded the rapid degeneration of mammalian nerves disconnected from their cell bodies. As degenerating axonal segments could be selectively stained, this
feature was widely exploited to identify the connections
among brain centers, thereby contributing to the general
acceptance of the unique trophic role of neuronal perikarya.
In those early times, the persisting survival of centrally
disconnected axons from crustacean nerves (30, 147) and
from a mouse mutant (212) was not known.
The concept of the exclusive trophic role of the
neuron soma gained a clearer meaning with the formulation of the basic dogma of molecular biology. The identification of DNA as the undisputed genetic repository of
protein primary structure, and of mRNA as its molecular
envoy, paved the way to the belief that neuron somas
were uniquely responsible for the synthesis of all axonal
and presynaptic proteins. The main foundation of this
hypothesis rested on the visualization of the Nissl substance in nerve cell bodies and large dendrites (87) but
not in axons. The Nissl substance was later identified as a
complex of riboliponucleoproteins using the newly developed cytospectrophotometric method (for a review, see
Ref. 45), and eventually as the ordered aggregate of free
and membrane-bound polysomes by EM analyses (248).
These key discoveries confirmed the high rate of protein
synthesis of the neuron cell body, and further supported
the view that axons were lacking protein-synthesizing
machinery, inasmuch as they lacked Nissl bodies. Curiously enough, an impressive number of authors referred
to the Palay and Palade paper as demonstrating the lack
of axonal ribosomes, notwithstanding its only dealing
with the ultrastructural analysis of neuronal cell bodies.
Presumably, such an unfounded claim was based on the
implicit consideration that the lack of the Nissl substance
reflected a lack of ribosomes, and therefore a lack of
protein synthesis!
The apparent absence of ribosomes in mature axons,
with the exception of the axon hillock and the nearby
axonal locations (337), was later established by conventional EM analyses (249, 257). This confirmed the already
prevailing opinion, irrespective of the caveat that this
method could identify subcellular particles only by their
nonspecific electron density. As a result, ribosome-like
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tiated neurons, local systems of gene expression are distributed throughout the axon domain. In this model, glial
cells synthesize and transcellularly deliver coding and
modulating transcripts to axons and nerve terminals
where these transcripts are eventually translated into proteins. Compared with the previous view, this mechanism
appears well tuned, efficient, and highly selective in the
spatial and temporal supply of the required gene products. It is also much less demanding on neuron metabolism. The experimental evidence supporting such a model
is described in the following sections.
However, it bears emphasis that this conceptual
framework does not exclude the role of the neuron cell
body in the modulation of local translation processes.
Several considerations suggest the contrary. From a general perspective, the correlation between cell body size
and the overall mass of neuronal processes (203) may be
taken to reflect such involvement. It should also be noted
that the fast delivery of soma-derived proteins and organelles to axons and nerve terminals (125) might contribute key controlling factors to local translation processes. Whether soma-imported proteins are directly involved in the regulation of local protein synthesis is yet to
be established. However, there has also been more direct
evidence for the transfer of cell body RNA to the axon:
1) the targeting of soma-derived BC1 RNA to the axonal
translation sites (periaxoplasmic plaques) of the goldfish
Mauthner axon (242), 2) the colocalization of ␤-actin
mRNA and its binding protein ZBP1 to the periaxoplasmic
plaques of goldfish and mammalian axons (287a), 3) the
targeting of soma-derived RNA-containing vaults to the
Torpedo nerve terminals (205), and 4) the demonstration
that, in well-differentiated Aplysia neurons, the synapsespecific long-term facilitation induced at a distal synapse
requires axoplasmic transport and somatic protein synthesis in addition to local protein synthesis (128).
On the other hand, earlier experiments designed to
verify the cell body origin of axonal RNAs using radiolabeled precursors consistently yielded controversial results (13, 34, 36, 106, 107, 160, 258, 263). More recently,
RNA granules were identified and characterized in neurons (reviewed in Ref. 171). The cell body origin of the
granules localized in the axon domain was demonstrated
in cultured or developing neurites (172, 197) but not in
mature axons (287; but see Refs. 205, 242). In fully differentiated neurons, soma-derived RNA granules appear to
be exclusively delivered to dendrites (141, 167, 222, 283).
The topic of the cell body origin of axonal and presynaptic
RNAs will be discussed in more detail in section V.
III. LOCAL PROTEIN SYNTHESIS IN AXONS
A major technical difficulty encountered in the experimental demonstration of protein synthesis in axons
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The dogma of an exclusive somatic origin of axonal
and presynaptic proteins was also weakened by two theoretical objections. One of them regarded the survival of
slowly transported axonal proteins, notably cytoskeletal
proteins, which would have required months or even
years to reach their distal-most targets. These expected
delays strongly clashed with the half-life of the transported proteins, which was not longer than a few days. As
a result, these proteins were bound to disappear from the
axon according to a proximo-distal gradient approaching
zero at relatively short distances from the neuron soma.
Such an expectation was clearly at odds with all available
evidence. The attempt to bypass this objection by the ad
hoc assumption that slowly transported proteins were
selectively endowed with longer half-lives (202) was
proven untenable by the demonstration that their average
half-life was not different from the accepted values (244,
245).
The second objection concerned the rapidly transported presynaptic proteins. If they originated exclusively
from the cell body, their participation in plastic events
would require short delivery times and a two-way signaling system operating between nerve terminals and neuron
soma. In view of the impressive terminal branching of
axons and of the thousands of its diverging synaptic connections, any presynaptic ending would need to instruct
the cell body to synthesize and send the set of proteins it
required, and concurrently provide the spatial coordinates required for a correct delivery. In turn, proteins
synthesized by the cell body would likewise be expected
to contain enough information to correctly guide them
through the multiple axonal branches they needed to
cross before reaching their targets (7, 120, 183).
Neither set of signals has been proven so far, and
signals of such complexity may hardly be compatible with
our present view of the neuron. Nonetheless, in an ingenious attempt to solve this problem, it was postulated that
activated synaptic terminals might be tagged by an unspecified local process not requiring protein synthesis.
Tagging would eventually allow the selective capture of
appropriate proteins synthesized by the cell body and
delivered to the entire neuronal cytoplasm (99). Such
mechanism has been proposed to explain long-term synaptic changes in activated synapses of Aplysia sensory
neurons in culture (44). Although plausible, this hypothesis has not received convincing experimental support in
other animal models. In addition, it does not seem to be
readily reconciled with the relatively frequent modifications of synaptic strength, nor with the possible concurrent requirement of different protein sets by different
nerve endings of the same neuron. Tagging would also
impose a heavy metabolic load onto the synthetic capacity of neuronal perikarya.
These two substantial objections may be readily resolved by the alternative possibility that, in well-differen-
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
derives from their strict association with glial cells. This
close anatomical relationship required that the translation
capacity of axons be distinguished from that of glial cells
by autoradiographic methods or microdissection procedures combined with microchemical analyses. A more
incisive alternative was offered by axons of unusually
large size, such as the giant axon of the squid and the
Mauthner (M-) axon of the goldfish, as they could be
analyzed with conventional biochemical methods.
A. Early Evidence
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Additional key observations were obtained following
the local application of actinomycin D to the central
stump of a DFP-treated and axotomized hypoglossal
nerve (176). Compared with the contralateral DFP-treated
and axotomized nerve, the level of AChE determined 4
days after axotomy was twice as high in the nerve treated
with actinomycin D. Comparable effects were present in
the intact nerve, irrespective of its previous treatment
with DFP. Moreover, in the untreated intact nerve, the
increment of AChE induced by actinomycin D was prevented by the local application of 5-fluororotate or puromycin. These results were interpreted to suggest that the
synthesis of nerve AChE was locally regulated at the level
of transcription by a repressor RNA whose synthesis was
blocked by actinomycin D. Comparable, seemingly paradoxical effects of actinomycin D had previously been
reported in the induction of liver enzymes (108).
Following the publication of these data, several
groups started to report the local incorporation of radioactive amino acids into axonal proteins of mammals,
amphibians, fish, and cephalopods, as described in the
following subsections.
B. Model Systems
The incorporation of radioactive amino acids into
axonal proteins was demonstrated in model systems under conditions excluding the contribution of nerve cell
bodies.
1. The squid giant axon
The unusually large size of this axon (diameter up to
0.5 mm or more) accounts for the relative ease with which
axoplasm may be extruded or axon may be internally
perfused. These features have long made it a favorite
object of neurophysiological studies (reviewed in Ref. 51)
and molecular biological investigations (97, 103, 114, 200,
266). The giant axon generally used is the most medial,
longest, and largest one of ⬃10 giant axons associated
with the corresponding number of stellate nerves (Fig. 2A).
The many cell bodies of each giant axon are segregated in
a special lobe of the stellate ganglion (the giant fiber
lobe). During development, the tiny axons of these neurons fuse together to give rise to the giant axon (225, 335).
Synthesis of axoplasmic proteins by isolated squid
giant axons (i.e., separated from their cell bodies) was
independently demonstrated by two research groups using different routes of administration of radiolabeled
amino acids. In one approach, the giant axon of the squid
Loligo pealii was incubated in artificial seawater containing a mixture of [14C]amino acids that were incorporated
into axoplasmic proteins at an approximately linear rate
for several hours (114). The proteins of the axon sheath
were synthesized at a markedly faster rate, as expected
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In the initial pioneering studies, the dogma of the
exclusive somatic origin of axonal proteins was tested
and found to be inadequate to explain the resulting observations. According to the cell body hypothesis (see
sect. II), the irreversible inactivation of a neuronal enzyme
would lead to its reappearance in the axon according to a
proximo-distal gradient (49, 184, 185). An experimental
protocol was then devised to implement the inactivation
of acetylcholinesterase (AChE) in cat cholinergic neurons
by the parental injection of diisopropylfluorophosphate
(DFP) (184, 185). However, at variance with expectations,
proximal and distal segments of hypoglossal and cervical
sympathetic nerves exhibited virtually equal rates of enzyme recovery after the elimination of AChE activity. As
the AChE inactivation by DFP was irreversible, the recovery of nerve AChE could not be due to reactivation of the
enzyme but rather to its de novo synthesis. The uniform
rate of AChE recovery in the hypoglossal nerve was confirmed even when the content of the perikaryal enzyme
was either substantially decreased by intracranial injections of DFP throughout the recovery period or kept
normal by selectively inactivating the nerve enzyme with
diisopropylphosphostigmine iodide, a compound unable
to cross the blood-brain barrier.
In a comparable series of experiments, these results
were confirmed using a more sensitive AChE assay (174).
In addition, they demonstrated that in untreated cats, 1
day after axotomy of the hypoglossal nerve, the level of
AChE markedly increased in the most distal portion of the
regenerating nerve stump. As this increase was inhibited
by puromycin, the effect reflected a local process of protein synthesis. A more conspicuous increment of AChE
was observed when the preexisting enzyme activity was
suppressed by DFP before axotomy. Under the latter
conditions, a previously decentralized nerve segment retained the capacity to synthesize AChE, as the enzyme
level significantly increased after an additional axotomy
of the decentralized stump. Interestingly, the latter effect
was completely blocked by the local application of either
RNase or 5-fluororotate, a compound interfering with
RNA synthesis. Likewise, the recovery of AChE in a DFPtreated intact nerve was prevented by the local administration of 5-fluororotate.
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from the presence of resident glial and connective tissue
cells. The synthesis of axoplasmic and sheath proteins
was strongly inhibited by puromycin and cycloheximide
(CXM), which indicated the involvement of a eukaryotic
translation system. In the other approach, the synthesis of
axonal proteins was demonstrated by microinjecting radiolabeled amino acids into giant axons of the squid
Dosidicus gigas (97). Interestingly, the stimulation of the
axon at 100 stimuli/s during the first 10 min of the 100-min
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FIG. 2. Schematic representation of the squid stellate ganglion and
nerves (A) and of the giant axon perfusion chamber (B). A: several nerve
bundles emerge from the ganglion in starlike fashion. Each nerve contains numerous small-diameter fibers and a single giant axon with large
diameter (up to 1 mm; shown in black). The giant axon is formed by the
fusion of many small axons originating from a corresponding number of
cell bodies segregated in the giant fiber lobe. B: following extrusion of
axoplasm, the giant axon is placed in the perfusion chamber containing
artificial seawater and [3H]uridine. The perfusing solution enters the
axon through a cannula and flows out in small droplets from its distal
end.
incubation period increased the incorporation about twofold. Conversely, when action potentials were abolished
by stretching or puncturing the axon, the incorporation
fell to negligible values.
In later studies, the synthesis of axoplasmic proteins
by the isolated giant axon of Loligo pealii was confirmed
(3), and the question of their cellular localization was
addressed (104, 200, 201, 277, 303). At the end of a series
of experiments, the conclusion was reached that the axoplasmic proteins synthesized by the isolated giant axon
were entirely derived from periaxonal glia (the glia-neuron protein transfer hypothesis). The hypothesis was entirely based on the presumed translational incapacity of
the axon, offering a sharp alternative interpretation of the
local synthesis of axonal proteins. The main observations
supporting this claim included the following: 1) extruded
axoplasm was unable to synthesize proteins, 2) newly
synthesized proteins accumulated in the perfusate of giant axons even when RNase was added to the internal
perfusing solution, and 3) most newly synthesized proteins were localized in the peripheral axon rim, as shown
by autoradiographic analyses (104, 200, 201, 303). These
data became one of the fundamental underpinnings of the
prevalent opinion rejecting the concept of axonal protein
synthesis. However, as detailed in a review article (112),
the same data allowed alternative interpretations, and did
not appear compelling with regard to the lack of proteinsynthesizing machinery in the giant axon.
An additional stronghold of the general disclaim of
local protein synthesis in axons was the demonstration
that squid giant axons only contained tRNA (31, 199). The
apparent lack of other cytoplasmic RNAs, notably rRNAs,
confirmed that axons could not synthesize proteins and
suggested that axoplasmic tRNA comprised only a few
species serving a different function such as addition of
activated amino acids to the native proteins (161). These
data were contradicted by two independent observations.
First, selective assays of single tRNAs and aminoacyltRNA synthetases demonstrated that the giant axon contained as many as six different tRNAs and the corresponding synthetases. In other words, all the tRNA species
assayed for were in the axoplasm (161). As these components could not be involved in other activities but translation processes, the findings contributed to tilt the balance in favor of axonal protein synthesis. Second, with
the use of microelectrophoretic methods, axoplasm was
shown to contain sizable amounts of rRNAs in addition to
an unusually large 4S RNA component and minor
amounts of other small RNAs (113). Furthermore, a number of independent analyses demonstrated that extruded
axoplasm also contained all the hundred species of soluble proteins and RNAs required for eukaryotic protein
synthesis, including initiation and elongation factors, and
all tRNAs and aminoacyl-tRNA synthetases (122).
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
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the axonal synthesis of NF proteins (M. Crispino, B. B.
Kaplan, and A. Giuditta, unpublished data).
The presence in the giant axon of all the components
needed for eukaryotic protein synthesis (113, 118, 119,
122, 161, 256) strongly suggested that active polysomes
were also present. This possibility was tested by incubating with [35S]methionine intact stellate nerves still connected to stellate ganglia and mantle muscle. Polysomes
purified from extruded axoplasm, axonal sheath and the
cell bodies of the giant axon were then displayed by
sedimentation on linear sucrose gradients (121). This experiment demonstrated that radiolabeled proteins from
each sample were almost exclusively localized in the
polysomal region of the gradient. In addition, pretreatment of polysomes with EDTA or RNase induced a drastic
shift of the radiolabeled proteins to the lighter regions of
the gradient, thus proving their nature of nascent peptide
chains. The latter data dissipated remaining skepticism
and represented the most compelling demonstration of
axonal protein synthesis.
The presence of polysomes in the squid giant axon
was also confirmed by ultrastructural analyses based on
electron spectroscopic imaging (ESI) methods (121) and
immunoelectron microscopy (284). In the ESI method,
substances containing phosphorus are selectively visualized as the electron beam impinging on phosphorus atoms
loses a certain amount of energy. Hence, in view of their
high phosphate content, ribosomes emit very strong signals (247). With ESI, large aggregates of polysomes were
found in the cortical axoplasmic layer, in close association with mitochondria, as well as in the axoplasm core
(33, 121, 224, 226). While the previous arguments against
the axonal presence of polysomes were based on negative
data, the ESI method and immunoelectron microscopy
provided definitive evidence for their presence. The translation of several axoplasmic mRNAs in the giant axon was
proved by RT-PCR analysis of purified axoplasmic polysomes. The list includes ␤-actin and ␤-tubulin mRNAs
(169) as well as the mRNA coding for the heavy chain of
kinesin (109).
2. The Mauthner axon
One of the first observations demonstrating the local
synthesis of axonal protein was obtained from the Mauthner (M-) neurons of the carpfish (Carassius carassius).
The large cell bodies of these two neurons are located on
either side of the rostral medulla from which their myelinated fibers decussate and course the entire length of
the spinal cord (78). With the use of autoradiographic methods, 1 day after the intracranial injection of [14C]lysine,
axons and myelin sheaths of M-fibers were found to contain radiolabeled proteins. Their distribution in the initial
2-mm axonal segment followed a proximodistal gradient
that moved distally and flattened out in the course of
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Final proof of axonal protein synthesis was nonetheless delayed until two additional sets of data became
available to demonstrate that the axoplasm of the giant
axon contained both a population of mRNAs and active
polysomes. The first piece of evidence was based on the
specific mRNA assay testing the capacity of purified RNA
to specify the synthesis of proteins in a cell-free rabbit
reticulocyte lysate supplemented with [35S]methionine
(251). Unexpectedly, when purified axoplasmic RNA was
used as a template and the translation products were
fractionated by SDS-PAGE, newly synthesized radiolabeled proteins were found to exceed 50 different species,
indicating that at least as many different translatable
mRNAs were present in the axoplasm (118, 119). The
mobility of the major bands corresponded to the axonabundant cytoskeletal proteins, such as actin, the two
tubulins, and neurofilament (NF) proteins. Notably, a few
radiolabeled axoplasmic proteins were missing in the
translation pattern of cell bodies RNA (and vice versa).
Substantial differences in the relative abundance of axoplasmic and cell body mRNAs were later detailed using
the method of competitive RT-PCR (48). In conclusion, at
variance with all expectations, the giant axon did contain
a large population of mRNAs that specified the synthesis
of proteins ranging from ⬃10 kDa to several hundred kDa,
some of which were absent in the cell bodies. Hence,
axoplasmic mRNA could not be considered the consequence of passive diffusion of cell body transcripts. These
data were confirmed and extended by kinetic hybridization assays of polyadenylated axoplasmic RNA with its
cDNA. This raised the estimated number of axoplasmic
mRNAs to at least 200 different species (256). Such a high
number was remarkable for an axon, even if kinetic hybridization analyses indicated that polyadenylated RNA
from the axon cell bodies displayed much greater sequence complexity, comprising several thousand mRNAs.
In situ hybridization assays established that poly(A)⫹
mRNA was distributed throughout the axoplasm of the
giant axon. These observations paved the way to the
preparation of the first cDNA library of axoplasmic
poly(A)⫹ mRNAs that allowed comparisons with a
cDNA library from the cell bodies (169).
Several clones of the axoplasmic library were identified by DNA sequencing, and the corresponding radiolabeled probes were used to confirm the presence of individual mRNAs in the axon by in situ hybridization. The list
included the mRNAs encoding ␤-actin and ␤-tubulin
(169), the heavy chain of kinesin (109), squid enolase (47),
and a polypeptide sharing partial homology with a calcium channel of the mammalian sarcoplasmic reticulum
(46, 326). In addition, using a riboprobe to mouse NF68
mRNA, the squid giant axon was shown to contain an
homologous NF mRNA (121). Later immunoadsorption
analyses with squid specific NF antibodies demonstrated
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into the M-axon of live fish (6). The process was sensitive
to inhibitors of protein synthesis (5). In these experiments, newly synthesized axonal proteins were ⬍5%
of those produced in the perikaryon if referred to unit
mass, but the value became 50-fold greater than in the
perikaryon when calculated in terms of overall axonal
mass. Protein synthesis appeared to occur in the axon
itself, as under these conditions myelin contained a much
lower amount of radiolabeled proteins. Interestingly, lowfrequency stimulation of the M-neuron increased axonal
protein synthesis twofold if the stimulation lasted 18 h,
but 4-h stimulation produced no effect. The enhanced
synthesis was no longer present 1 day later (90). A comparable effect had been previously reported in frog sciatic
nerves subjected to high-frequency stimulation (213). The
significant decrease in protein content of rat sciatic
nerves after in vitro electrical stimulation or following a
period of prolonged swimming (163) suggests that the
turnover of axonal proteins is a local process.
The M-axon has also been a model system in studies
of the components of the protein synthesis machinery. As
mentioned earlier, the most important single reason to
consider the axon incapable of protein synthesis was the
alleged absence of ribosomes, as determined by conventional EM methods. This view only regarded mature axons, as ribosomes were detected in developing axons (40,
294). Hence, it was generally believed that ribosomes
disappear as the axon matures (150) and, as a corollary,
that the mature axon is devoid of rRNA and mRNA (31,
199). A pivotal turn drastically revising these views took
place when RNA was identified in the adult axons of
goldfish, cat, crustaceans, and squid. In these axons, the
concentration of RNA was consistently much smaller
than in the cell bodies, but its total content was several
times higher in the total axonal mass (9, 76, 83, 85,
126, 175).
Axonal RNA was initially detected and characterized
by microdissection of fixed M-axons (76, 83). In microchemical analyses, the average RNA content of goldfish
(Carassius auratus) M-axon corresponded to 0.15 pg/
␮m. As a result, in a 4-cm axon, total axonal RNA
amounted to 6,000 pg, while the cell body only contained
2,000 pg. As expected, RNA was highly concentrated in
the perikaryon (1% wt/vol). RNA was also present in the
myelin sheath, with 30% higher amounts than in the axon.
Interestingly, axon and myelin RNA shared a similar base
composition, suggesting a common origin. Nonetheless,
the adenine/guanine (A/G) ratio was slightly but significantly higher in the myelin (0.6) than in the axon (0.56),
irrespective of the distance from the cell body. Both
compartments contained a substantial amount of a nucleotide fraction rich in A, raising the possibility that they
might contain some mRNA.
Several RNA species were eventually identified in the
unfixed M-axons by electrophoretic fractionation (179).
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several days. Hence, proteins synthesized in the cell body
appeared to move into the proximal axon at a rate of
0.1–3.0 mm/day. Interestingly, smaller but significant
amounts of radiolabeled proteins were also detected in
the more distal regions of axon and myelin sheath
throughout the 4-cm length of the M-fiber as early as 1 h
after [14C]lysine administration. This quick appearance of
radiolabeled proteins could hardly be attributed to the
axonal flow. The concentration of these proteins markedly increased after 3 h, and their distribution did not
follow a rostrocaudal gradient. Myelin was consistently
more labeled than the axon at all times. The latter data
suggested that a local system of protein synthesis was
present throughout the M-fiber.
Strong support for this interpretation was obtained
by incubating spinal cord segments with [35S]methionine
(78). Under these conditions, isolated M-axons and myelin sheaths contained radiolabeled proteins, whose synthesis could not be attributed to the cell bodies that were
dissected away with the medulla. The local synthesis of
M-fiber proteins was strongly inhibited by puromycin,
which indicated the involvement of a local ribosomal
system. With the use of a similar experimental protocol,
the newly synthesized proteins of the M-axon were eventually fractionated by gel electrophoresis, and the major
bands were shown to correspond to the axon-enriched
cytoskeletal proteins, including actin, ␣- and ␤-tubulins,
and NF proteins. Their synthesis was strongly inhibited by
CXM (181). Interestingly, the DNA-binding transcription
inhibitor actinomycin D markedly decreased the incorporation of radiolabeled leucine, lysine, or an amino acid
mixture in the M-fiber (79). The data implied that a large
part of axonal protein synthesis requires local DNA templates localized in glial nuclei, and indicated the marked
metabolic instability of the transcribed mRNAs. These
observations were in general agreement with the high
RNA turnover of the M-axon (77, 162).
Isolated M-fibers containing myelin but not glial cell
bodies were still capable of synthesizing proteins at a
linear rate for several hours. Synthesis of the new protein
was partly sensitive to puromycin and acetoxycycloheximide, but virtually insensitive to actinomycin D and to
the bacterial translation inhibitor chloramphenicol (CAP)
(78). Under these conditions, one-fourth of the radiolabeled proteins were present in the axon, in agreement
with the comparable distribution in M-fibers dissected
from incubated spinal cord pieces, in which glial cell
bodies were still present. The lack of CAP inhibition
suggested the involvement of an extramitochondrial
translation system. This suggestion was confirmed by the
prevalent recovery of the newly synthesized radiolabeled
proteins of isolated M-fibers in the microsomal and cytosol fractions (81).
The local synthesis of M-axon proteins was later
confirmed by microinjection of a radiolabeled amino acid
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
Physiol Rev • VOL
RNP (173) and implicated in protein synthesis (296),
PARPs may serve as axonal translational centers targeted
by the involved RNAs.
The cortical zones of the M-axon and mammalian
root axons also contain 7S RNA, a key component of the
signal recognition particle (SRP) that correctly addresses
the synthesis of secretory and integral membrane proteins
(316). Using immunocytochemical methods, the SRP 54
protein has been shown to colocalize with PARPs in
rabbit ventral root fibers (297). These observations suggest the intriguing possibility that axons may be able to
synthesize secretory or integral membrane proteins, even
if the Golgi apparatus has not been documented in axons.
Evidence supporting the axonal synthesis of this class of
proteins has been reported in decentralized cultured snail
axons microinjected with the conopressin receptor mRNA
(289).
C. Vertebrate Axons
In the newt Triturus viridescens, 45 min to 18 days
after the intraperitoneal injection of [3H]histidine, the
radiolabeled amino acid was detected in brachial nerves
examined by light and EM autoradiography (281). Silver
grains were localized in Schwann cell, myelin lamellae,
and axons. According to a semi-quantitative analysis, the
level of radioactivity was highest in Schwann cell bodies
and myelin during the first few hours, but progressively
increased in the axon. Interestingly, the intensity and
pattern of the radiolabeled signals were not affected by
decentralizing the nerve, or by incubating dissected
nerves with the radioactive precursor. The latter observations and the presence of silver grains throughout the
length of the nerve suggested that the data were due to
the activity of a local system of protein synthesis.
As described in section IIIA, the most incisive and
productive line of research on axonal protein synthesis
started with the demonstration of AChE resynthesis in cat
nerves (174, 176, 184, 185). Soon after, a small amount of
RNA was identified in the axons of cranial nerve roots of
adult cats (175). The concentration of this RNA was only
0.2% of that in the neuron soma, and its A/G ratio was 0.57
just like in the M-axon. Not much later, axonal protein
synthesis was directly demonstrated in the rabbit accessory spinal roots incubated with [3H]leucine. In these
experiments, the labeled amino acid was incorporated
into the proteins of microdissected axons (177). The incorporation proceeded at an almost linear rate for ⬃8 h,
after an initial lag period of ⬃2 h. The lack of CAP
inhibition excluded the involvement of bacteria and mitochondria and suggested the participation of a eukaryotic ribosomal system. As prolonged preincubation with
actinomycin D did not modify the rate of protein synthesis, the involved mRNAs appeared to be metabolically
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They included a prevalent 4S RNA, cytoplasmic 26S and
18S rRNA, and minor amounts of unidentified 15S and 8S
components that were not present in myelin. As also
noted for squid (113), the 4S peak of M-axons included
other small RNAs, such as a 7S and a 5S RNA. The relative
content of these small RNAs was markedly higher than in
the M-cell bodies. This unusual abundance was tentatively
attributed to the need to maintain a sufficiently high concentration of tRNAs throughout the axoplasm to ensure
optimal rates of protein synthesis for polysomes residing
in restricted axonal domains. With time, the apparent lack
of morphologically identifiable ribosomes led to a patent
discrepancy with the evidence of eukaryotic rRNA in
large model axons (76, 80, 113, 179). The apparent absence of axonal ribosomes was likewise contradicted by
the local cytoplasmic synthesis of proteins. Thus, for
those supporting the axonal location of a protein synthetic machinery, axonal ribosomes were assumed to be
in an occult form. A number of hindering conditions were
thought to be responsible for the difficulty of detecting
them by conventional EM methods, such as their limited
number or their association with unusual structures.
The puzzle of vertebrate axons containing rRNA but
not allowing the visualization of ribosomes (80, 179) was
eventually resolved by staining whole mounts of native
M-axons with YOYO-1, a nucleic acid probe that becomes
highly fluorescent when it binds to RNA (186). With the
use of this procedure, RNase-sensitive fluorescent structures containing more intensely fluorescent “puncta”
were identified at the surface of M-axons and other goldfish spinal nerve fibers. When examined by phase-contrast
or differential interference contrast microscopy, these
newly defined structures (periaxoplasmic plaques or
PARPs) protruded from the axon surface and were distributed at irregular intervals along the entire axon. As
shown by colabeling with rhodamine-conjugated phalloidin, PARPs were embedded in the peripheral F-actin
cytoskeleton. With ESI, the fluorescent puncta present
inside the plaques and in the underlying axoplasm were
identified as polysomal aggregates. This conclusion was
confirmed with immunocytochemistry using ribosomespecific antibodies (186).
The presence of individual mRNAs in M-axons (e.g.,
the mRNA of the medium-sized NF protein) was demonstrated by RT-PCR methods (324), while in situ hybridization showed that ␤-actin mRNA is preferentially distributed in goldfish and mammalian PARPs. Myosin Va and
kinesin II proteins also coincide with PARPs (285). Besides these molecular motor proteins, axoplasmic plaques
appear to contain BC1 RNA, a brain-specific transcript of
RNA polymerase III. Following its microinjection in the
M-cell body, radiolabeled BC1 RNA was detected in the
M-axon (and in M-dendrites) where it was intermittently
located according to the characteristic PARP distribution
pattern (242). Because BC1 RNA is associated with a 10S
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Physiol Rev • VOL
in Schwann cell cytoplasm, myelin sheath, and in between
the inner myelin lamella, as well as in the axon. The
identification of the newly synthesized light NF protein by
immunoadsorption and two-dimensional gel electrophoretic methods demonstrated that the corresponding
mRNA was translated in the nerve. These observations
were consistent with the CXM-sensitive synthesis of NF
proteins in axons of rat spinal roots and in M-axons (181).
Altogether, these results suggested that some transcripts
encoding neuron-specific proteins such as NF may be
transferred from glia and translated in the axon.
This idea was further supported by other investigations using in situ hybridization and EM methods. The
mRNAs of the medium and light NF proteins were unexpectedly detected in the Schwann cells of young rats
examined under a variety of conditions, including differentiation (170), demyelination, or unilateral nerve transection (271). In the latter condition, the light NF mRNA
was also detected in the contralateral nerve, implying that
it also occurred in normal nerves. Medium and light NF
mRNAs were also transiently expressed in Schwann cells
resident in the distal stump of lesioned sciatic nerves
during axon degeneration and remyelination of ingrowing
axons. Interestingly, translation of these mRNAs was random and unpredictable in Schwann cells, as shown by
immunocytochemical analyses (95). These results further
support the idea that NF transcripts are not intended for
translation in glial cells but are rather destined to be
transported and translated in regenerating axons. Identification of NF mRNA sequences in the Schwann cell
cDNA library further confirmed that some neuron-specific
proteins are transcribed in glial cells (170).
Most recently, the modulation of protein synthesis in
the sciatic nerve was neatly demonstrated by RNA interference methodology (240). According to this work, adult
mouse sciatic nerve contains several protein components
of the RNA-induced silencing complex (RISC), including
Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase. Hence, the
result extended the previous findings that the same modulatory complex is present in neurons (189), in developing
axons and growth cones (135), and in dendrites (276).
Interestingly, local application of short-interfering RNAs
(siRNA) against neuronal ␤-tubulin triggered RISC formation, and specifically produced marked decrease in both
mRNA and protein levels of ␤-tubulin in the nerve fibers
(Fig. 3). The reduction of axonal tubulin content was
confirmed by the significant decrement in retrograde axonal transport assessed by reduced fluorogold labeling of
spinal motoneurons. The existence of such a siRNA-based
regulatory mechanism in the axon raises the question of
whether this mechanism might be used by the periaxonal
glial cells to selectively modulate the synthesis of axonal
proteins.
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stable. In a later study, electrophoretic fractionation of
axonal proteins synthesized in vitro by rat spinal ventral
roots showed that axon-enriched cytoskeletal proteins
are among the newly synthesized proteins, such as actin,
tubulins, and NF proteins (181). This pattern was similar
to that of the nerve cell bodies, but differed from the
pattern of newly synthesized myelin proteins. In all cases,
the synthesis of these proteins was markedly inhibited by
CXM. The distinctive pattern of newly synthesized myelin
proteins indicated that a local system of protein synthesis
is also present in myelin. As myelin represents the overwhelming cytoplasmic periphery of the Schwann cell, its
local system of protein synthesis may play similar roles to
those played by the axon translation system with respect
to the nerve cell body.
The key data demonstrating a local increase of AChE
in the proximal stump of axotomized cat hypoglossal
nerves (174, 176, 184, 185) prompted further studies of
this effect. In the axotomized hypoglossal nerve of the
rabbit, in vitro analyses of [3H]leucine incorporation into
axonal proteins revealed a dramatic burst of protein synthesis in a narrow axonal segment proximal to the lesion
site (298, 299). When the data were normalized with the
concurrent accumulation of proteins transported by the
axoplasmic flow, the peak of protein synthesis occurred
18 h after axotomy, marking a 20-fold increase. The specific activity of the newly synthesized proteins was consistently higher in the axon than in the myelin, as the
latter did not exhibit a significant response to axotomy.
Interestingly, in analogy with the axonal synthesis of
AChE (see sect. IIIA), this striking effect was still present
in the decentralized nerve stumps (299). CAP was not
inhibitory, but CXM strongly inhibited axonal protein synthesis in both intact nerves and axotomized nerve tips.
Microelectrophoretic fractionation of the proteins synthesized by the latter axonal segment indicated that they
were substantially different from newly synthesized proteins released in the incubation medium (98). Hence,
protein synthesis in the decentralized nerve was independent of the cell body. This important set of observations
further supported the existence of protein synthesis in the
myelinated mammalian axons and its transcription modulation by periaxonal glial cells.
Local protein synthesis was also studied in sciatic
nerve, the biggest nerve in the body of mammals. In vivo
incorporation of locally applied radiolabeled amino acids
in the axons of lesioned and unlesioned rat sciatic nerves
was demonstrated by autoradiographic methods (25, 53).
More recently, with the use of RT-PCR methods, intact
and axotomized sciatic nerves of adult rats were shown to
contain the mRNAs encoding all NF proteins. In addition,
the content of the light NF mRNA was higher in the
proximal stumps of axotomized nerves than in control
nerves (286). Interestingly, in situ hybridization analyses
demonstrated that the light NF mRNA was also localized
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
Axonal mRNAs were also found in the mammalian
CNS. Notably, studies with rats revealed that several
mRNAs encoding peptide hormones of the posterior hypophysis were present in mature axons of the hypothalamic vasopressin and oxytocin neurons (165, 166, 232,
233, 300, 301). Under conditions of functional load, the
content of some axonal mRNAs increased much more
than in the neuron cell bodies. Furthermore, following the
upregulation of the galanin peptide elicited by salt loading, preprogalanin mRNA was identified in neurohypophysial axons (198). Besides the transcripts for the hormones, the mRNA encoding the light NF protein was also
detected in the axons of the same neurons (232). Ribosomes have not been reported in neurohypophysial axons
of control rats, but they are present in the hypertrophic
vasopressin-containing axons following chronic stimulation
by a salt-loading regime (223). In addition, the mRNAs encoding olfactory receptors and the olfactory marker protein
were identified in the axons of olfactory receptor neurons
(70, 268, 307, 328).
Following the demonstration of PARPs in the cortical
layer of M-axons (see sect. IIIB2), analogous formations
were identified in axons of rabbit and rat ventral root
fibers stained by YOYO-1 and antiribosomal antibodies
(187). These structural correlates were likewise protrudPhysiol Rev • VOL
ing from the axon surface, and the fluorescent puncta
were identified as polysomal clusters by ESI analyses
(Fig. 4, A–D). Compared with M-axon, the mammalian
PARPs are quite thin (⬃1 ␮m) and occur with variable
length (usually ⬃10 ␮m) at irregular intervals along the
axon (from 10 to 100 ␮m; average 25 ␮m). Most PARPs
are located at internodes. In view of their large variability
in size and morphology, PARPs are likely to represent a
dynamic biological structure that changes its location and
distribution along the axon, in agreement with the dynamic features of the F-actin layer in which they are
embedded (293). The F-actin cytoskeleton may be involved in the assembly and distribution of mRNAs and
ribosomes in the axon, as it does in other cells (21, 139,
279). In support of the functionality of cytoskeleton-anchored ribosome and mRNA, elongation factor 1 was
found to be bound and regulated by F-actin (52, 208).
F-actin may be also involved in the radial displacement
of axonal components that are longitudinally transported
along microtubules. Although more has to be known
about the precise biological role of the mammalian periaxoplasmic plaque, its identification as a ribosome-containing axonal structure has made an important contribution to the hypothesis of axonal protein synthesis. Interestingly, the presence of ribosomes in periaxoplasmic
domains was witnessed ante litteram more than a decade
ago. In an EM study of myelinated axons of rabbit dorsal
root ganglia, ribosomes were mainly distributed in the
cortical axoplasm of a few axons. Some ribosomal aggregates were associated with ER (250; Fig. 4E). This observation is one of the few direct EM evidences for the
presence of axonal ribosomes in mature neurons, foreshadowing the discovery of PARPs.
The existence of a local system of protein synthesis
in mature mammalian axons is now generally accepted
(reviewed in Ref. 262), as also demonstrated by studies
dealing with its participation in nerve regeneration. The
requirement of the local synthesis of proteins for axonal
sprouting was initially shown in mouse sciatic nerve regenerating in vitro (75) and later confirmed in vivo. In a
live rat, the regeneration of the crushed peroneal nerve
was markedly reduced by the local application of CXM, as
elongation of the axonal sprout was reduced by 58% during the 3-day recovery period (101). This effect occurred
irrespective of the absence of live Schwann cells, confirming the axonal localization of the system of protein synthesis.
The axonal synthesis of proteins is crucial in nerve
regeneration. In an incisive experiment, the neurite extension of NGF-differentiated PC12 cells was shown to depend on the translation of a short-lived mRNA encoding
the ribosomal L4 protein (302). Administration of antisense oligonucleotides for L4 protein inhibited neurite
outgrowth in both PC12 cells and in adult rat sensory
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FIG. 3. Marked decrease of neuronal tubulin mRNA and protein in
mouse sciatic nerve treated with anti-tubulin siRNA. A: nerve fibers
treated with a specific anti-tubulin siRNA. B: nerve fibers treated with an
unspecific siRNA. Blue/purple signals indicate the presence of ␤-tubulin
mRNA visualized by in situ hybridization; the red fluorescence corresponds to the immunodetection of ␤-tubulin by TUJI antibodies. White
arrows, localization of fluorescent signals within nerve fibers. [Modified
from Murashov et al. (240).]
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GIUDITTA ET AL.
neurons cultured after a crush to the sciatic nerve. Further studies of the latter preparation demonstrated that
regenerating axons contain L4 and other ribosomal proteins, translation factors, rRNA and mRNA, and are capable of protein synthesis (339). Blocking protein synthesis
led to a rapid retraction of growth cones, provided that
axonal sprouts were separated from cell bodies by either
axotomy or colchicine-based inhibition of axonal transport. Hence, axonal synthesis of protein appears crucial in
maintaining growth cones and other intrastructure of regenerating axon. Translation factors, ribosomal proteins,
and rRNAs were also detected in motor axons of ventral
spinal roots examined 7 days after in vivo lesion of the
peripheral nerve.
The role of axonal protein synthesis in regeneration
was also highlighted by the presence of proteins with
nuclear localization signals (importins) in the distal regions of axons. Axonal ␤-importin increases after nerve
lesions by the local translation of its encoding mRNA. The
complex of ␤-importin and dynein with other axonal proteins leads to its retrograde transport into the neuron
soma that enables the central response to axonal regeneration (133, 134). Forty proteins that were retrogradely
transported have been identified by two-dimensional
PAGE and mass spectrometry in regenerating axons of
the snail Lymnea stagnalis. Several of them comprise
posttranslationally modified proteins and calpain cleavage products of a 51-kDa intermediate filament protein
(254, 255). Similar analyses made on purified axons of
injury-conditioned dorsal root ganglion neurons led to the
Physiol Rev • VOL
identification of cytoskeletal proteins and a large number
of other proteins, including heat shock proteins, endoplasmic reticulum proteins, enzymes, and proteins associated with neurodegenerative diseases (330). Interestingly, RT-PCR analyses of the same preparation indicated
that regenerating axons also contain mRNAs encoding
these proteins, suggesting their translation in the axon.
Treatment of regenerating axons with nerve growth factor
(NGF) or brain-derived neurotrophic factor (BDNF)
markedly enhanced the content of axonal mRNAs encoding cytoskeletal proteins, as a result of their increased
rate of delivery via axoplasmic transport. No significant
effect regarded other axonal mRNAs.
Further insights into the mechanism of the Wallerian
degeneration of decentralized axons were provided by
analyses of the Wlds mutant mouse which undergoes a
markedly delayed degeneration. The protective mutant
gene encodes a chimeric protein resulting from the fusion
of nicotinamide mononucleotide adenyltransferase-1
(NAD synthetase) with the NH2-terminal fragment of the
ubiquitination assembly factor Ube4b (216). In Drosophila, this mouse protein suppresses self-destruction signals originating in transected axons and activating Draper
engulfment glial receptors that mediate the elimination of
injured axons by glial cells (146, 215). The extensive
changes in glial morphology elicited by lesioned axons
through the upregulation of these glial receptors did not
occur in Draper mutants. As a result, decentralized axons
were not eliminated from the nervous system. Interestingly, the Wlds protein remained ineffective in the clearing
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FIG. 4. Axonal ribosomes and polysomes in rabbit nerve fibers. A–D: periaxoplasmic ribosomal plaques in isolated axon whole mounts from
rabbit ventral root myelinated fibers. Low-power differential interference contrast micrographs (A) and the corresponding phase-contrast images
(B) show that compact integral formations (plaques) are periodically distributed in the cortical zone of the axon. The colocalization of these
structures with ribosomal domains is shown by the correspondence of the phase images (C) with the immunofluorescent labeling of rRNA by the
Y-10B anti-rRNA monoclonal antibody (D). Scale bars, 10 ␮m. [Modified from Koenig et al. (187).] E: conventional EM micrographs of myelinated
axons from sensory spinal nerves. Some ribosomes are attached to a membrane of an endoplasmic reticulum tubule (top panel, arrows) or are close
to a mitochondrion; ribosomal clusters are also close to the endoplasmic reticulum or to mitochondria (bottom panels). S, Schwann cells. [Modified
from Pannese and Ledda (250).]
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
of supernumerary axons during the development of flies
and mice, although Draper receptors and the ubiquitinproteasome system were needed to implement both injury-induced and development-induced axon degeneration
(for reviews, see Refs. 23, 331). Altogether, these studies
imply that periaxonal glial cells may play an important
role in the physiology of degenerating and regenerating
axons.
D. Other Axons
E. Neurons in Culture
Protein synthesis was clearly demonstrated in regenerating axons of the cultured ganglion cells from goldfish
retina (182). Axons severed from the original explant
without extraneous cells incorporated radiolabeled
amino acids at an approximately linear rate for 3 h. Intact
axons exhibited similar rates. This incorporation did not
reflect bacterial or mitochondrial protein synthesis as it
was inhibited by CXM and emetine, but not by CAP.
Microelectrophoretic fractionation of the proteins synthesized in the axonal fields indicated that most radioactivity
Physiol Rev • VOL
was associated with ␤-tubulin while actin and other proteins were labeled to a lesser extent. In contrast, ␣-tubulin
was not labeled (180). In the intact preparation, the depolarizing condition induced by 100 mM KCl was more
inhibitory to protein synthesis in the axonal fields than in
the ganglion cell bodies, in analogy to the comparable
effects reported for the squid giant axon and its cell
bodies (252). Local synthesis of ␤-tubulin and ␤-actin
was also demonstrated in intact growing axons of rat
sympathetic ganglion cells using immunoabsorption
methods (88).
Axons of cortical neurons from embryonic rat brain
(22) and chick sympathetic neurons (246) contain poly(A)⫹
mRNA, as shown by in situ hybridization or RT-PCR methods. In the latter study, mRNA associated with granules
was present at axon branch points, varicosities, and
growth cones, but ␣-tubulin mRNA was absent, in agreement with data derived from growing axons of goldfish
retinal explants (181). mRNAs coding for the Tm-5 tropomyosin isoform and the SCG10 protein were identified in
the growing axons of developing rat cerebellar neurons or
PC12 cells (131, 132). SCG10 is believed to be an actinbinding protein that modulates membrane-cytoskeletal
interactions.
In cultured neurons derived from rat brain, growth
cones contain polyribosomes, as shown by EM analyses
(24). In addition, using in situ hybridization methods,
axonal granules and growth cones were found to contain
␤-actin mRNA. The association of these granules with
microtubules suggests that ␤-actin mRNA is transported
along these cytoskeletal elements. The selectivity of this
process was highlighted by the lack of ␥-actin mRNA in
the axonal granules, in analogy to the lack of ␣-tubulin
mRNA in the axons of sympathetic neurons (246).
In an elegant experiment with cultured neurons of
the snail Limnea stagnalis, a heterologous mRNA microinjected into isolated neurites was translated into its encoded protein, the egg-laying hormone (305). These observations were the first to demonstrate the axonal translation of a single exogenous mRNA. In later work, isolated
snail axons plated in cultured dishes were microinjected
with the heterologous mRNA coding for the conopressin
receptor (289). One day later, the receptor protein was
identified in the microinjected axons by electrophysiological and immunocytochemical methods, showing that this
axon-made protein is functional. As the conopressin receptor is coupled to G protein, the depolarizing response
elicited by conopressin was successfully blocked by
the nonhydrolyzable analog guanosine 5⬘-O-(2-thiodiphosphate) (GDP␤S).
For a detailed discussion of the differences between
growing and mature axons, readers are referred to Alvarez et al. (7).
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The presence of axonal RNA was also demonstrated
in other animal models. In the crayfish Procambarus
clarkii, the concentration of RNA in giant axons is 0.02%
(wt/vol), compared with 0.22% in periaxonal glia and 5.7%
in neuronal perikarya (9). Accordingly, in a 4-cm axon
containing 1.67 pg RNA/␮m, the total amount of RNA
corresponds to 67,000 pg, compared with ⬃1,000 pg in the
cell body. Crayfish axonal RNA is largely extramitochondrial. The different CG/AU ratios of axonal, glial, and
perikaryal RNAs (0.53, 0.77, and 0.97, respectively) were
attributed to the relative rRNA content of these tissue
samples. Autoradiographic analyses of nerve chords incubated with tritiated uridine showed that newly synthesized RNA was initially present in Schwann cell nuclei,
but became progressively more concentrated in Schwann
cell cytoplasm and axons with longer incubation times. In
the axon, most radiolabeled RNA was localized near the
axolemma, notably in the vicinity of abutting Schwann
nuclei, suggesting its derivation from periaxonal Schwann
cells.
In the slowly adapting stretch receptor neurons of
the lobster (Homarus agammarus), RNA was also detected in the axons at a concentration of 0.06%, while the
concentration of cell body RNA was 0.5% (85, 126) (see
also sect. VIIIB1). More recently, in the snail Lymnaea
stagnalis, ultrastructural in situ hybridization methods
demonstrated that axons and nerve terminals of the neurons secreting the egg-laying hormone contained the corresponding mRNA in secretory granules (72).
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IV. LOCAL PROTEIN SYNTHESIS
IN NERVE TERMINALS
A. Model Systems
1. Squid nerve endings
FIG. 5. Ribosomes and polysomes in the presynaptic terminals of the squid photoreceptor neurons. A: schematic representation of the retinal
photoreceptors. While the cell bodies are located in the retina, and the axons in the optic nerve, the large “carrotlike” nerve terminals are located
in the outer plexiform layer of the optic lobe. Hence, the three subcellular domains are anatomically segregated. B: EM micrograph of the
synaptosomal fraction of the squid optic lobe. Large presynaptic synaptosomes are predominant and easily identified by their content of synaptic
vesicles and mitochondria (scale bar, 1 ␮m). C: electron spectroscopic imaging (ESI) of the large optic lobe synaptosomes showing bright light
signals due to the phosphorus-rich ribosomes and polysomes. White arrows, synaptic vescicles. [Assembled from Martin et al. (226) (A), Crispino
et al. (57) (B), and Crispino et al. (58) (C).]
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Nerve terminals can be enriched in subcellular fractions containing synaptosomes, which are plasma membrane-enclosed structures derived from synapses. When
protein synthesis was demonstrated in the mammalian
synaptosomes (see sect. IVB), their heterogeneous composition drew some doubts on the nature and origin of the
synaptosomal translation system. While most studies attributed the presence of a cytoplasmic (and a mitochondrial) system of protein synthesis to nerve terminals (14,
329), outlandish explanations were advanced with regard
to the nature of the synaptosomal protein synthesizing
machinery, or claims were made of its prevalent or almost
exclusive localization in postsynaptic and/or glial elements. Valid arguments against these positions will be
addressed in the following pages. Nonetheless, the heterogeneous composition of mammalian synaptosomes still
allows controversial interpretations of the data. Some of
the problems raised by their diverse cellular origin have
been solved by studies of the model synaptosomal fraction of squid optic lobes.
The axons of squid retinal photoreceptor cells innervate the cortical layer of the optic lobe where they make
synaptic contacts with the resident amacrine neurons
through unusually large, carrotlike nerve endings (Fig. 5A).
Upon homogenization of the optic lobe, these nerve endings give rise to large presynaptic synaptosomes (Fig. 5B),
which are the most abundant components of the synaptosomal fraction. As the optic lobes do not harbor additional nerve terminals of comparable size (50, 336), large
synaptosomes of squid optic lobe are exclusively derived
from the nerve terminals of photoreceptor neurons.
Investigations of this unique preparation provided
conclusive evidence that nerve endings contain active
systems of cytoplasmic and mitochondrial protein synthesis (26, 56 –58, 73, 137, 226). At variance with the mammalian preparations, the synaptosomes of cephalopod optic lobes are obtained as a floating layer by their differential centrifugation in slightly hypotonic sucrose homogenates.
This preparative method leads to better preservation of the
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
Physiol Rev • VOL
by Ca2⫹ may be in large part funneled through protein
kinase C (PKC) and its downstream targets, eIF-2 or
eEF-2. The data suggest that incidental surge of Ca2⫹ at
the nerve endings (such as elicited by ischemic or other
degenerative insults) may have an additional deleterious
consequence. When presynaptic protein synthesis is inhibited by high levels of cytosolic Ca2⫹, the repressed
local synthesis of nuclear-encoded mitochondrial proteins may lead to the reduced supply of mitochondrial
energy. Consequently, further inhibition of presynaptic
protein synthesis may follow. Such a feedback loop might
eventually cause degeneration of the nerve terminal and
possibly lead to “dying back” of the related axonal segment.
2. Aplysia nerve endings
In elegant experiments with cultured Aplysia neurons, the long-term potentiation of a sensory-motor synapse was subjected to a series of detailed manipulations
to elucidate the underlying molecular mechanisms. In the
intact animal, this synapse is responsible for the facilitation of the gill withdrawal reflex elicited by nociceptive
stimuli. The experiments regarded cultured sensory neurons endowed with a bifurcated axon establishing separate synapses with two different motor neurons. As longterm facilitation (LTF) may be mimicked in vitro by repeated spaced administration of serotonin to the sensory
nerve terminals, such treatment at one synapse induced
LTF in that synapse, but not in the other synapse. These
experiments demonstrated the great spatial selectivity of
LTF, and the essential role of presynaptic protein synthesis in allowing LTF. Indeed, local protein synthesis at the
sensory nerve terminal increased threefold in the potentiated synapse but not in the control synapse. In addition,
local inhibition of presynaptic protein synthesis completely abolished LTF (44, 220, 221). Interestingly, transcription and translation events triggered in the cell body
by serotonin administration to the presynaptic sensory
terminal were also essential for LTF. This suggested the
activation of the somatic gene expression system by retrograde signals and the delivery of newly synthesized
somatic gene products to the same terminal.
Comparable data demonstrating the importance of
both somatic and local protein synthesis in synapse-specific LTF were more recently described in well-differentiated Aplysia ganglia mediating the syphon withdrawal
reflex (128). These authors demonstrated that in a distal
sensory-motor synapse a similar interplay between somatic and local systems of protein synthesis was necessary to allow LTF. The involvement of axonal transport
was proven by the nocodazole block of LTF due to its
disruption of axonal neurotubules. Nonetheless, the large
distance between the synapse and the cell body (2–3 cm)
induced strains in the attempt to fit the known rates of
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synaptosomes, and may in part be responsible for their
high rate of protein synthesis (57, 137). In most other
respects, squid synaptosomes resemble mammalian synaptosomes.
The essential purity of squid optic lobe synaptosomes was first suggested by the electrophoretic pattern
of their newly synthesized proteins, which substantially
differed from those of neuronal cell bodies or glial cells
(56, 57). The low level of contaminating structures was
confirmed by light and EM autoradiographic analyses,
which also demonstrated the nearly exclusive localization
of newly synthesized proteins in large synaptosomes (58).
Polysomes purified from optic lobe synaptosomes incubated with [35S]methionine were shown to contain nascent peptide chains (57), and to be free from microsomal
polysomes (110). Polysomes were likewise identified in
the large optic lobe synaptosomes using ESI (Fig. 5C) (58)
and in the carrotlike nerve endings of intact squid optic
lobes using ESI and YOYO-1 fluorescent staining (226).
Proteins synthesized by optic lobe synaptosomes or
by purified synaptosomal polysomes include several
members of the NF protein family (56), as well as calexcitin, a learning-related protein (93). RT-PCR analyses of
synaptosomal polysomes have led to the identification of
mRNAs coding for cytoskeletal and ribosomal proteins,
translation factors and, most notably, mitochondrial proteins encoded by nuclear DNA (110, 111). The association
of these mRNAs with purified polysomes indicates that
the corresponding proteins are synthesized in presynaptic
synaptosomes. These data were confirmed and extended
using two-dimensional electrophoretic methods that demonstrated the presynaptic synthesis of ⬃80 different protein species, several of which were identified by mass
spectroscopy (164). They include cytoskeletal proteins,
enzymes, novel proteins, and mitochondrial proteins encoded by nuclear DNA. The synthesis of mitochondrial
proteins in nerve terminals has physiological significance,
as it adds a new dimension to the concept of presynaptic
autonomy. Indeed, presynaptic (and axonal) mitochondria can no longer be considered exclusively dependent
on nuclear-encoded mitochondrial proteins derived from
the distant neuron soma, as these proteins are locally
synthesized.
Protein synthesis in presynaptic synaptosomes may
be under the control of cell signaling pathways, as shown
by its strong modulation by cytosolic Ca2⫹. With the use
of intracellular and extracellular Ca2⫹ ionophores, chelators, and other modulators, protein synthesis of optic lobe
synaptosomes was shown to proceed at nearly optimal
rates when the intraterminal Ca2⫹ level was maintained
within the normal physiological range. When this level
increased or decreased, protein synthesis was markedly
depressed (26, 27, 57). As strong inhibition was also induced by calphostin, a selective inhibitor of protein kinase C (26), modulation of presynaptic protein synthesis
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B. Vertebrate Nerve Endings
Nonmitochondrial organelles originally detected in
the mitochondrial fraction of rat brain (259) were eventually purified by sedimentation on sucrose density gradients and largely identified as pinched-off nerve terminals (synaptosomes) on the basis of their content of synaptic vesicles and mitochondria (67, 127). Since then,
mammalian brain synaptosomes purified with different
protocols have been found to contain other minor particulates such as free mitochondria and fragments of axons,
dendrites, and glial processes (54, 105, 124, 136, 234, 264,
Physiol Rev • VOL
310; for a review, see Ref. 329). In parallel with these
observations, synaptosomes were examined for their capacity to synthesize proteins, as previously proposed for
axons (184, 185). These investigations were prompted by
the unusually high rate of protein synthesis displayed by
brain mitochondria that otherwise appeared to share the
same features of liver mitochondria (43). An important
clue emerged when protein synthesis by the brain mitochondrial fraction was unexpectedly found to be inhibited
by CXM, in addition to CAP (334). The data implied that
the brain mitochondrial fraction contained nonmitochondrial structures capable of cytoplasmic protein synthesis.
The suggestion that these structures were synaptosomes came from experiments with rat brain slices incubated with a radiolabeled amino acid (14). The subcellular
fractionation of the radiolabeled slices indicated that the
specific activity of synaptosomal proteins increased linearly with time from the very beginning of the incubation.
The absence of a lag period, also lacking in vivo (74; for a
review, see Ref. 18), suggested that newly synthesized
synaptosomal proteins originated locally. This interpretation was proven correct by the demonstration that purified synaptosomes catalyzed the incorporation of a radiolabeled amino acid into protein (235–237). The reaction
did not require addition of exogenous soluble factors, was
insensitive to RNase, and was partially inhibited by CAP
and more strongly inhibited by puromycin or CXM. The
translation products were associated with most synaptosomal subfractions except synaptic vesicles. Newly synthesized soluble and membrane proteins were selectively
inhibited by CXM, while CAP only blocked the synthesis
of mitochondrial proteins. On the whole, the data indicated that, in addition to a mitochondrial translation system, synaptosomes contained cytoplasmic polysomes and
all the soluble factors and energy sources required for
their protein synthetic activity. Comparable results were
obtained with synaptosomes purified on a discontinuous
Ficoll gradient (1, 196). They appeared to be better preserved and to exhibit an optimal rate of protein synthesis
in the presence of 100 mM NaCl and 10 mM KCl (15).
Because protein synthesis was not affected by addition of
ATP, soluble factors, and RNase, it was attributed to
membrane-enclosed synaptosomes rather than to contaminating microsomal polysomes. Likewise, a major contribution of bacterial and mitochondrial sources was excluded by the partial inhibition exerted by CAP. On the basis
of EM analyses, the origin of most Ficoll synaptosomes
(60%) was attributed to nerve terminals, while most other
structures appeared to be postsynaptic elements rarely containing ribosomes.
Ultrastructural autoradiographic analyses further established the subcellular origin of Ficoll synaptosomes
harboring newly synthesized proteins. According to Cotman and Taylor (54), almost half of the radiolabeled
proteins were present in synaptosomes of presynaptic
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retrograde transport, somatic transcription and translation, and anterograde transport into the time frame allotted by the progressive cell body-dependent stabilization
of LTF. Among several alternative explanations, the authors mentioned “different mechanisms could mediate
LTF at mature, intact synapses, compared with mechanisms at newly formed synapses in dissociated cell cultures.” These mechanisms might regard the intercellular
interplay of the presynaptic terminal with the perisynaptic glia.
Local presynaptic protein synthesis is also needed
for synapse formation between cultured neurons. Removal of the cell body of Aplysia presynaptic sensory
neurons and of postsynaptic target motor neurons does
not interfere with the formation of synaptic connections.
Under these conditions, synaptic efficacy increases for 2
days and is reversibly blocked by anisomycin, an inhibitor
of protein synthesis (274). Isolated sensory synapses deprived of their cell bodies are still able to express a form
of LTF that is dependent on local protein synthesis and is
even greater than that produced when the cell bodies are
present. However, this form of LTF decays after 1 day,
thereby showing the relevance of the cell body for longerterm memory (210). Changes in synaptic efficacy are associated with the accumulation at presynaptic sites of the
sensory neuron-specific neuropeptide sensorin and sensorin mRNA. Selective interference with sensorin mRNA
by dsRNA does not decrease the level of presynaptic
sensorin but prevents LTF, suggesting the need for its
local translation (214, 275). The target-dependent synthesis and release of sensorin acting on autoreceptors mediates the increase in synaptic efficacy (148). These effects
also require the activation of multiple protein kinases and
their complex interactions (149, 209).
On the whole, these data demonstrate that local
translation processes are absolutely required for implementing long-term plastic changes on the synapses established between cultured neurons or well-differentiated
neurons. They also raise intriguing questions on the complex interactions between somatic and local gene expression processes.
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
Physiol Rev • VOL
size (8.9 kDa) and of large size (⬎60S). Upon electrophoretic fractionation, synaptosomal poly(A)⫹ RNA manifested a clearly distinctive distribution, with a marked
prevalence of high-molecular-weight components. In addition, the 7S RNA component of the synaptosomal
poly(A)⫹ RNA fraction was much less evident in the
mitochondrial fraction, and altogether absent in microsomes. The selective synaptosomal localization of 7S RNA
and of a larger RNA component was confirmed in adult
rats (71). In that paper, intrasynaptosomal mitochondria
were reported to contain cytoplasmic rRNAs and 4S RNA,
while synaptic vesicles contained 5S RNA, 4S RNA, and
cytoplasmic rRNAs. The cytosol fraction only contained
4S RNA. Hence, synaptosomes define a unique cytoplasmic microdomain whose RNA compositional profile is
clearly distinguishable from that of other cell fractions.
It is of interest that in experiments on young rats,
brain synaptosomes and mitochondria were reported
to contain a much higher proportion of poly(A)⫹ RNA
than polysomes (⬃18% compared with 7%), and to display
a similar capacity to direct protein synthesis in a cell-free
translation system (69). Synaptosomal membranes and
synaptosomal mitochondria had a similar content of poly
(A)⫹ RNA (15.5 and 13.5%, respectively), but the capacity
to direct protein synthesis in a cell-free system was much
lower for the RNA prepared from synaptosomal membranes. Lower concentrations of poly(A)⫹ RNA were reported in synaptosomes and mitochondria from adult
rats, but their contents were still higher than in microsomes (60). Altogether, these observations may reflect
that the synaptosomes generated by nerve endings may
have higher activity of protein synthesis in the developing
brain of younger animals.
The presence of particular RNA species in nerve
endings was also documented in other systems. RNP particles known as “vaults” have been reported to be highly
enriched in the cholinergic nerve terminals of the electric
organ of Torpedo marmorata in which they are closely
associated with synaptic vesicles (138). Vaults are transported along the axon in either distal or proximal directions, as indicated by their accumulation on either side of
a nerve lesion (205). Vault RNA is a small RNA transcribed by RNA polymerase III.
1. Alternative interpretations: a commentary
The evidence described above supported the view
that nerve endings contained a prevalent cytoplasmic system of protein synthesis and a less active mitochondrial
system. Nonetheless, the validity of this concept was
repeatedly challenged, and alternative interpretations
were proposed to account for the data. To simplify our
task, these interpretations were assigned to two groups
according to whether 1) the existence of presynaptic
protein synthesis was accepted but attributed to unusual
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origin, while most other newly synthesized proteins were
localized in unidentified structures, some of which might
be also derived from nerve endings. In addition, about
one-tenth of the radiolabeled proteins were associated
with membrane-bound particles containing endoplasmic
reticulum. Comparable data were reported by Gambetti
et al. (105). According to these authors, up to half of the
radiolabeled proteins were present in synaptosomes originating from presynaptic endings. Of these proteins, onefifth were associated with mitochondria and the rest were
localized in the periphery of presynaptic synaptosomes.
Of the remaining newly synthesized proteins, 5% was
present in fragmented bodies, ⬃10% in nonpresynaptic
structures without ribosomes, and about one-third in nonpresynaptic structures containing ribosomes. The latter
particulates were attributed to fragmented glial cells or
postsynaptic elements that displayed a sixfold higher labeling than presynaptic synaptosomes. Hence, the rat
synaptosomal fraction contains other protein-synthesizing cell elements besides the predominant presynaptic
nerve endings.
Inhibitors of glycolysis, tricarboxylic acid cycle, and
oxidative phosphorylation strongly inhibited synaptosomal protein synthesis, which proved its dependence on
ATP generation by intrasynaptosomal mitochondria (15,
236). Likewise, lowering the osmolarity of the incubation
medium markedly decreased synaptosomal protein synthesis, due to synaptosome swelling and rupture of the
plasma membrane, with the consequent release of synaptosomal contents into the medium. The dependence of
synaptosomal protein synthesis on the appropriate concentrations of Na⫹ and K⫹ in the medium was traced to
the activity of the synaptosomal membrane Na⫹-K⫹ATPase, in agreement with the marked inhibition by
ouabain, a specific inhibitor of the ion pump (10, 15, 308).
Ouabain inhibited both CXM- and CAP-sensitive systems
of synaptosomal protein synthesis, and its inhibition was
mediated by lowering the level of intrasynaptosomal
K⫹ (309).
The synaptosomal fraction contains extramitochondrial cytoplasmic RNA and other elements of the protein
synthesis machinery. In early studies (14), cell body mitochondria and intrasynaptosomal mitochondria from rat
brain were shown to contain similar concentrations of
DNA (6.3 ␮g/mg protein) and RNA (12–13 ␮g/mg protein),
but higher concentrations of RNA were present in synaptosomal membrane fractions (up to 21 ␮g/mg protein). In
later studies, extramitochondrial synaptosomal RNA was
identified as rRNAs and 4S RNA that were consistently
attributed to contaminating elements (59, 69, 89, 211).
Nonetheless, in addition to mitochondrial RNAs and typical cytoplasmic rRNA (18S, 28S) and small RNA (4S, 5S),
the synaptosomal fraction of adult rat brain contained
two RNA components that were absent in mitochondrial
and microsomal fractions (59, 60). They were of medium
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Physiol Rev • VOL
(see sects. III and IV), the opinion that mammalian nerve
endings are substantially devoid of protein synthesis
seems to be still lingering. The ongoing controversy on
this issue has been reviewed elsewhere (7, 120, 183, 262).
2. Effects of stimulation on synaptosomal
protein synthesis
Chemical and electrical stimulation of rat synaptosomes have been reported to induce marked increments
in protein synthesis, but most effects have been ascribed
to postsynaptic elements, often by default. Depolarizing
treatments of the membrane potential elicited a marked
enhancement of synaptosomal protein synthesis. Electrical stimulation of synaptosomes induced a fourfold increment of protein synthesis accompanied by increased O2
uptake (322). The largest increments regarded SDS-soluble and SDS-insoluble proteins of the “junctional complex,” while less conspicuous effects concerned Triton
X100-soluble proteins. In addition, several proteins with
molecular masses between 10 –18 and 32– 87 kDa became
more intensely labeled. These effects were markedly inhibited by tetrodotoxin, a Na⫹ channel blocker. On the
contrary, protein synthesis in C-6 glioma cells was not
modified by the same electrical stimulation, suggesting
that the enhancement of synaptosomal protein synthesis
was not mediated by the translational machinery of glial
origin. As the increment of protein synthesis was repressible with CAP more than with CXM, the electric stimulation appears to affect the mitochondrial protein synthesis
more than the cytoplasmic counterpart.
A different method was used to examine the effect of
a depolarizing medium (40 mM KCl) on a synaptosomal
fraction prepared by filtration of brain homogenates
through pores of decreasing size (323). Under these conditions, the content of polysomes rapidly but transiently
increased. Stimulation of protein synthesis in this mode
persisted in the presence of CAP, thus excluding a mitochondrial contribution, but was strongly repressed by
BAPTA-AM, a chelator of cytosolic calcium. The stimulating effect of depolarizing media was mimicked by glutamate, PKC-activating agent, and agonists of metabotropic
glutamate receptors (mGluR). The PKC inhibitor calphostin C strongly inhibited the activation of mGluR and consequently repressed protein synthesis in rat synaptosomes, supporting the idea that Ca2⫹ signaling and mGluR
are implicated in the synaptic protein synthesis.
A rather unusual approach was followed to distinguish neuronal and glial contributions to the changes in
protein synthesis in the rat sensory-motor cortex induced
by electrical stimulation of the brachial plexus in vivo.
The approach was based on the assumption that glucose
is the preferred substrate of neurons while acetate is the
preferred substrate of glial cells (2). After an intraperito-
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types of protein synthesizing machinery, or 2) the existence and role of presynaptic protein synthesis was marginalized in the belief that most synaptosomal protein
synthesis derived from dendrites and/or glial processes.
Followers of the first alternative assumed that synaptosomal protein synthesis was due to either 1) a unique
CAP-resistant, CXM-sensitive system selectively present
in brain mitochondria and/or synaptosomes; 2) an aberrant CAP-sensitive extramitochondrial system; or 3) a
nonribosomal incorporation of amino acids. However,
these proposals were based on hypothetical speculations
rather than on sound evidence, were mutually exclusive,
or were quite limited in scope. In addition, they ignored
the wealth of consistent data demonstrating the coexistence of cytoplasmic and mitochondrial systems of protein
synthesis in synaptosomes. The detailed description of
these data and the related criticism were provided in a
comprehensive review (see Ref. 7).
If we now consider the second group of papers marginalizing the existence of presynaptic protein synthesis,
they appear to be based on a misrepresentation of pivotal
EM autoradiographic data which actually demonstrate
the localization of a major share of newly synthesized
proteins in presynaptic synaptosomes (54, 105; see also
sect. IVB). Instead, their attention was focused on ribosome-containing synaptosomes assumed to derive from
dendritic processes or glia (264, 291, 310). In the paper by
Verity et al. (310), most protein synthesis was assumed
not to be associated with presynaptic endings. The argument was based on the observation that the radiolabeled
proteins of denser synaptosomal subfractions displayed
higher specific activities. However, the substantial amount
of total radioactive proteins recovered in the lighter fractions derived from the presynaptic endings was not taken
into account. Hence, the negligible contribution of nerve
endings to synaptosomal protein synthesis appears to be
an overstatement. With that said, it should be made clear
that the presence of postsynaptic and glial elements in
mammalian synaptosomal preparations cannot be denied.
We merely object to the conclusion that synaptosomes
generated by those nonpresynaptic structures represent
the main source of newly synthesized synaptosomal proteins. In a previous detailed review article (7), we fully
acknowledged the experimental evidence supporting the
presence of glial (136, 278) and dendritic elements (264,
323) in mammalian brain synaptosomes.
In summary, while there is general agreement that
mammalian synaptosomes contain a cytoplasmic system
of protein synthesis in addition to a mitochondrial system,
the conclusion that protein synthesis is absent or negligible in nerve endings does not withstand critical analysis.
Rather, this notion appears more based on the reiteration
of the long-standing dogmatic view than on the available
data. Despite the convincing demonstrations that the
axon domain contains a local system of protein synthesis
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
3. Mitochondrial protein synthesis in
synaptosomal fractions
The mitochondrial synthesis of proteins examined in
synaptosomal fractions of the rat cerebral cortex is most
active in 10- to 13-day-old rats, but soon declines after
weaning and remains essentially unmodified in 2-yr-old
animals (211). Synthesis of mitochondrial proteins did not
correlate with the content of corresponding mitochondrial mRNAs. Indeed, the ND3 and ND5 peptide subunits
of NADH dehydrogenase were nearly absent among newly
synthesized mitochondrial proteins, despite the normal levels of their mRNAs. Accordingly, gene expression in synaptosomal mitochondria appears to be largely regulated
at the translation level. Gel electrophoretic fractionation
of translation products of synaptosomal mitochondria
demonstrated their strong inhibition by CAP. Surprisingly, CXM also inhibited the synthesis of a few proteins
with molecular mass below 50 kDa that are generally
considered mitochondrial proteins. Hence, it appears
that, in the rat brain synaptosomal fraction, an extramitochondrial system of protein synthesis is at work utilizing a selective subset of mRNAs. This apparently paradoxical effect can be explained by recalling that, in squid
presynaptic synaptosomes, nuclear-encoded mitochondrial proteins are synthesized by the local cytoplasmic
system of protein synthesis (110, 164).
4. Nerve endings of the Mauthner neuron
Only a limited amount of work has concerned the
short axon collaterals of the M-axon and their terminal
projections. Nonetheless, these elements have been
shown to contain polysomes using ribosome-specific antibodies (see Fig. 14 of Ref. 7), and more recently ESI
methods (R. Martin and E. Koenig, unpublished observations). In these nerve terminals, most ribosomal aggregates display a peripheral localization.
Physiol Rev • VOL
V. SYNTHESIS OF AXONAL AND PRESYNAPTIC
RNA: THE CELL BODY HYPOTHESIS
The identification of cytoplasmic RNA in the axon
domain of vertebrate and invertebrate species raised the
question of its cellular origin. Initial views held, almost by
default, that axonal RNA derives from the neuron cell
body, in agreement with the influential concept of axonal
transport of proteins and organelles (125). However, the
experimental demonstration of this hypothesis by radiolabeling perikaryal RNA and monitoring its displacement
toward distal axonal sites was met by considerable difficulties in the interpretation of the data. Indeed, at variance with the axonal transport of radiolabeled somatic
proteins, the wave of radiolabeled RNA invading the
nerve was generally associated with, and often preceded
by, a wave of radiolabeled RNA precursors (16, 34, 36,
106, 107, 160, 258, 263). Under these circumstances, the
RNA precursors entered periaxonal glia and were incorporated into radiolabeled RNA that was retained in the
glial cells and partly transferred to the axon. Hence, axonal RNA might be produced locally as well as being
transported from the cell body. Because of this experimental pitfall, axonal transport of RNA has been clearly
demonstrated only in a few model systems, such as the
transport of 4S RNA in regenerating goldfish axons (160).
Nonetheless, in more recent experiments with welldifferentiated neurons, the microinjection of radiolabeled
BC1 RNA into the cell body of the goldfish M-neuron was
followed by its anterograde axonal transport (242). In
addition, the microinjection of truncated fragments of this
small RNA indicated that the 5⬘-segment specifically targeted it to the axon. The experiment also showed that
BC1 RNA colocalizes with PARPs (Fig. 6A). More recently, fish and mammalian PARPs have been shown to
also contain ␤-actin mRNA and its binding protein ZBP-1
(287a). As the latter protein allows the association of
mRNAs to the molecular motors of the kinesin family
accountable for anterograde axonal transport, PARP’s
mRNAs may be derived from the neuron cell body. The
axonal transport of soma-derived small RNAs is likewise
indicated by the accumulation of RNA-containing vault
particles on the proximal side of a Torpedo nerve crush.
As shown in Figure 6B, vault particles start to increase
linearly a few hours after the crush, while the linear
accumulation of synaptic vesicles does not display a lag
period. This different behavior may be interpreted to
indicate that the distal transport of vault particles in
well-differentiated neurons only occurs after the lesioninduced neuronal dedifferentiation is under way. An analogous, albeit more tentative, explanation might hold for
the axonal transport of BC1 RNA in the goldfish M-neuron, provided a comparable process of dedifferentiation is
assumed to have followed the microinjection of this RNA
into the neuron soma.
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neal administration of [14C]glucose, newly synthesized
proteins increased significantly in the stimulated cortex,
while administration of [14C]acetate resulted in minor
decrease. Interestingly, most enhanced protein synthesis
took place in the synaptosomal fraction following
[14C]glucose administration (35). Despite the fact that
glucose can also be used by glia, the lack of stimulating
effect by acetate favors the interpretation that synaptosomal protein synthesis is stimulated by neuronal activity.
Local protein synthesis has been demonstrated to be
crucial to the BDNF-induced long-term potentiation
(LTP) of hippocampal synapses (168, 338), but no distinction was made as to the presynaptic or postsynaptic localization of the protein synthesis.
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VI. LOCAL SYNTHESIS OF AXONAL RNA
A. Model Systems
Neuronal mRNAs are largely associated with cytoskeletal elements, notably microtubules and actin filaments (139, 279, 290). As part of RNA granules also containing ribosomes and proteins, these mRNAs were later
shown to move distally along microtubules in the neurites
of cultured neurons (172, 197). Highly compacted RNA
granules have been isolated and characterized from cultured neurons of embryonic rat forebrain (190) and from
adult mouse brain (167, 141). These granules are prevented from engaging in translation activity during transport, but attain a less compact organization and give rise
Physiol Rev • VOL
1. The squid giant axon
Evidence for the local synthesis of axoplasmic RNA
was first obtained when microinjected radiolabeled uridine was incorporated into RNA in the giant axon of the
squid Dosidicus gigas (96). Experiments testing the possible origin of axoplasmic RNA from the neuron cell
bodies or from periaxonal glia were later made on the
giant axon from Loligo vulgaris (62). With the use of an
intact stellate ganglion-nerve preparation placed into an
elongated chamber, the ganglion was confined at one end
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FIG. 6. Evidence of anterograde axonal transport of RNA. A: radiolabeled BC1 RNA microinjected in the cell body of mature goldfish
Mauthner neurons was transported into the dendrites and the axon.
Following the surge of radioactivity in the axon, radiolabeled RNA that
selectively associated with axonal periaxoplasmic ribosomal plaques
(arrowhead) was attributed to soma-derived BC1 RNA. [Modified from
Muslimov et al. (242).] B: RNA-containing presynaptic vault particles
were accumulated in adult Torpedo electric nerves. Movement of vault
particles was monitored by the accumulation of the major vault protein
MVP100 (}) on the proximal side of a crush made on the elecric nerve
of Torpedo marmorata. The transport of the synaptic vesicles SV2
protein (䊐) was also determined. [Modified from Li et al. (205).] Note
that the accumulation of the presynaptic SV2 protein is already evident
1 h after the crush, while that of the vault MVP100 protein is negligible
for the first 3 h. The delayed accumulation of the latter protein suggests
that the anterograde transport of RNA-containing vault particles might
be triggered by the axon reaction elicited by the nerve crush, rather than
reflecting a natural occurrence in native unlesioned nerves.
to active polysomes upon depolarization (190). RNA granules from mouse brain contain as many as 42 proteins and
are translocated by kinesin exclusively into the dendritic
domain (141, 167, 222, 283). Neuronal RNA granules have
been distinguished into three classes on the basis of their
macromolecular composition, i.e., transport RNPs, stress
granules, and P (processing) bodies (171). Stress granules
and P bodies are, respectively, involved in recruiting
mRNAs to regulate their translation or to degrade them.
So far, stress granules and P bodies have only been identified in dendrites, and transport RNPs have only been
detected in growing neurites and in mature dendrites. To
our knowledge, none of these three types of RNA granules
has yet been described in the axons of well-differentiated
neurons (287, 317; but see Ref. 205).
The necessary participation of somatic translation
processes in plastic events occurring at synaptic sites has
been initially reported in cultured Aplysia neurons (44,
220) and, more recently, in well-differentiated Aplysia
neurons (128). In the latter preparation, the synapse-specific long-term facilitation induced at a distal synapse
required active protein synthesis at the presynaptic site
and at the neuron soma, as well as axoplasmic transport.
The latter two requirements strongly suggest that the
neuron soma is involved in the stabilization of the synaptic plastic change but fall short of suggesting that this
event requires the delivery of soma-derived transcripts to
the nerve terminal.
In conclusion, despite the clear demonstrations in
cultured neurites and developing axons, only a few available data support the contribution of soma-derived neuronal transcripts to the process of gene expression in the
axons and nerve endings of differentiated neurons. Hopefully, this putative intraneuronal pathway will be more
extensively investigated in the future to elucidate the
nature of the cell body’s participation to peripheral plastic
events vis à vis with the concurrent local contribution of
periaxonal and perisynaptic glial cells.
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
RNA manifested a much larger proportion of low molecular weight components compared with the RNAs synthesized in the periaxonal glial sheath or the nerve cell
bodies (229).
With periaxonal glia being the sole possible origin of
the locally synthesized RNA in the isolated axon, the
experiments with squid giant axons were eventually continued with internally perfused axons (Fig. 2B), which
allowed more direct kinetic analyses of RNA synthesis.
With the collection of perfusate samples at intervals, radiolabeled RNA was shown to enter the axon perfusate
within minutes after the addition of [3H]uridine to the
incubation chamber. The radiolabeled RNA continued to
accumulate in the perfusate at an approximately linear
rate for several hours. This process was markedly inhibited by the transcription inhibitor actinomycin D. In sedimentation analyses on linear sucrose gradients, most
newly synthesized perfusate RNAs were of low molecular
weight, some of them colocalizing with a tRNA marker. In
addition, sizable amounts cosedimented with marker subribosomal particles (92, 117). The prevalence of low molecular weight RNAs was in agreement with similar observations made with isolated squid giant axons (229).
The essentially linear accumulation of radiolabeled
RNA in the perfusate was exploited to further examine
the mechanism of glial RNA synthesis and transfer (62,
265). In a large set of experiments, the perfused axon was
subjected to a number of manipulations after the basal
rate of radiolabeled RNA delivery to the axon perfusate
stabilized. Initial tests aimed at reproducing the modifications of glial membrane potential elicited by high-frequency stimulation of squid and crayfish giant axons that
were attributed to the release of one or more neurotransmitters (91, 207, 312, 313). These experiments led to the
identification of several receptors on the glial plasma
membrane, including a cholinergic receptor and metabo-
FIG. 7. Axoplasmic RNA synthesized by isolated squid giant axons includes tRNA and rRNAs. Isolated giant axons and giant fiber lobes were
incubated 8 h with [3H]uridine. A and B: electrophoretic fractionation of radiolabeled RNA purified from the giant fiber lobes. C and
D: electrophoretic fractionation of radiolabeled RNA purified from the extruded axoplasm of giant axons. A and C show the presence of 4S tRNA
and of 20S and 31S rRNAs. B and D confirm the presence of 4S tRNA and demonstrate the disassembly of 31S rRNA into its 20S and 17S moieties.
Electrophoretic migration is toward the left. Vertical arrows mark the position of native squid tRNA and rRNAs. [Modified from Rapallino et al.
(265).]
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sealed off from the nerve by silicone grease. To determine
if axoplasmic RNA was synthesized in the neuronal cell
bodies, radiolabeled uridine was added to the ganglion
compartment, and incubation was allowed to proceed up
to 24 h. Despite an extensive series of analyses, newly
synthesized axoplasmic RNA did not appear to be derived
from the neuron cell bodies, as it only reached the most
proximal short segment of the axon that also contained
large amounts of the radiolabeled precursor. Conversely,
incubation of isolated nerves with [3H]uridine gave unequivocal evidence of the local synthesis of axoplasmic
RNA (62). Its amount was sizable after 4 h and markedly
increased after 8 h presumably because radiolabeled RNA
synthesized in periaxonal glial cells needed additional
time to reach the axoplasmic core. The glial origin of
axoplasmic RNA was also confirmed in vivo. One day
after [3H]uridine was injected near the stellate nerve of
live squid, radiolabeled RNA was recovered from the
axoplasm of axon segments near the injection site, while
the neuronal cell bodies manifested no evidence of incorporation of the injected RNA precursor.
Axoplasmic RNAs synthesized by isolated giant axons
were eventually identified as tRNA and rRNAs by electrophoretic fractionation (Fig. 7), and as poly(A)⫹ RNA by
adsorption on oligo(dT)cellulose (265). rRNAs were identified by the unique features of squid rRNAs, whose larger
species (30S) is formed by the noncovalent association of
the smaller component (20S) with an rRNA of lower size
(17S) (41). As these bonds are easily broken, in a significant number of analyses newly synthesized axoplasmic
RNA displayed 20S and 17S species rather than 30S and
20S rRNAs. Newly synthesized axoplasmic poly(A)⫹ RNA
represented 1% of the total radiolabeled RNA. Axoplasmic
RNA synthesized by isolated squid giant axons was also
shown to be assembled in ribonucleoprotein particles. At
short incubation time, newly synthesized axoplasmic
535
536
GIUDITTA ET AL.
beled RNA to the axon perfusate, implicating the glial
NMDA receptor in the picture (Fig. 8D). Similar effects
were observed when the glial nicotinic cholinergic receptor was activated by carbachol. In addition, when antagonists of either NMDA (MK801) or cholinergic receptors
(D-tubocurarine) were used in combination with the corresponding agonists, the delivery of radiolabeled RNA to
the axon perfusate remained at its basal level. Hence, the
stimulating effect of the neurotransmitters on glial RNA
transfer is specific to the receptors. Together with the
previous data (62, 229, 265), the above results provide
convincing evidence that the transfer of glia-synthesized
RNA into the axoplasmic domain is modulated by axonglia communication (92, 117). Comparable cell-to-cell
RNA transfer takes place in different eukaryotic cells,
such as the follicular cells and the oocytes of insects and
reptiles (29, 217, 238).
A key problem raised by the intercellular transfer of
glial RNAs and RNPs to the axons (see sect. VI, A2 and B)
or other neuronal domains (see sects. VIIB and VIIIB) regards the mechanism of such intercellular translocation
process. Comparable problems are raised in all cases of
transcellular exchange of macromolecules or organelles
in eukaryotic organisms. Most likely, the underlying
mechanisms may be different in different systems. Starting with the cytoplasmic bridges joining the follicular
cells and the oocyte (217, 238) or glial cells and crayfish
axons (253), a number of different mechanisms have been
proposed. They include an invagination of glial cytoplasm
into the axon followed by pinching off and engulfment of
the glial invagination (103, 195), or a combined process of
trans-endocytosis leading to the formation of spinules
(288). More recently, a novel mechanism has been documented in different cell types including cultured PC12
nerve cells. The mechanism involves the intercellular formation of 50- to 200-nm diameter membrane-bound nanotubules containing F-actin and myosin Va. This specialized intercellular structure was postulated to mediate the
3
FIG. 8. Glial modulation of [ H]RNA delivery to the axon perfusate. After the basal rate of RNA transfer was established, experimental
manipulations were applied (vertical arrows). Marked increments in the content of perfusate [3H]RNA are induced by the following treatments.
A: addition of 100 mM KCl to the incubation medium leading to massive depolarization of both axonal and glial plasma membranes. B: perfusion
of the axon with a low concentration of K⫹ that induces depolarization of the axon membrane only. C: addition of N-acetylaspartylglutamate (NAAG)
to the incubation medium. D: addition of N-methyl-D-aspartate (NMDA) to the incubation medium. NAAG activates a glial metabotropic glutamate
receptor and generates glutamate and N-acetylaspartate by ectopeptidase hydrolysis. Glutamate (but not N-acetylaspartate) also activates a glial
ionotropic glutamate receptor (NMDA receptor). [Modified from Eyman et al. (92).]
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tropic and ionotropic glutamate receptors. Their activation
was responsible for a long-lasting glial hyperpolarization
(⬃10 mV) from the resting value of about ⫺40 mV. This
condition was mimicked by exposing the perfused squid
giant axon (and its surrounding glia) to 100 mM K⫹ that
depolarized all plasma membranes. Interestingly, this
treatment shifted the basal linear kinetics of radiolabeled
RNA accumulation in the axon perfusate by creating a
marked biphasic increase of the RNA transfer rate
(Fig. 8A) (117). Essentially similar results were obtained
with the selective depolarization of the axon plasma membrane attained by drastically lowering the concentration
of K⫹ in the internal perfusing solution (Fig. 8B). Altogether, these data raised the possibility that the delivery
of radiolabeled RNA to the axon perfusate may be modulated by axon-released neurotransmitters activating glial
receptors.
This suggestion was strongly supported by the effects
produced by agonists of the glial glutamatergic and cholinergic receptors. One such agent, the dipeptide N-acetylaspartylglutamate (NAAG) had been shown to be the first
neurotransmitter released by stimulated crayfish axons
(102, 304). Addition of NAAG to the incubation medium
also produced a dual wave of enhanced delivery of radiolabeled RNA to the axon perfusate, mimicking the effect
of membrane depolarization (Fig. 8C). Comparable results were obtained by addition of glutamate, one of the
products of ectopeptidase-mediated hydrolysis of the axon-released NAAG. On the contrary, no effect was yielded
by N-acetylaspartate, the other product of NAAG hydrolysis that had been shown not to alter glial potentials.
Both NAAG and glutamate bind to class II metabotropic
glutamate receptors, but only glutamate binds to the glial
NMDA receptor (102, 304). Hence, similar experiments
were made with NMDA and D-aspartate, an additional
NMDA receptor agonist that is present in the axoplasm of
squid giant axons (65). Both NMDA and D-aspartate produced comparable increments in the delivery of radiola-
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
translocation of organelles and membrane proteins
(143, 273).
2. The Mauthner axon
537
RNA synthesized by isolated M-fibers remains a bit more
puzzling even if a mitochondrial derivation appears likely.
B. Vertebrate Axons
Physiol Rev • VOL
Following the demonstration of low concentrations
of axonal RNA in the cranial nerve roots of adult cats
(175), evidence of the local synthesis of mammalian axonal RNA was provided in rabbit accessory nerve roots
(177). In these experiments using 3-h incubation with
radiolabeled RNA precursors, the synthesis of axonal
RNA was markedly inhibited by actinomycin D, but most
newly synthesized RNA was resistant to RNase, presumably because it was shielded by a dense cytoskeletal
matrix (187). Pulse-chase experiments with cat nerve
roots suggested that the axonal RNA originated from an
exogenous source (178), presumably periaxonal cells.
The local synthesis of axonal RNA was also indirectly
suggested by analyses of RNA content and base composition of spinal motoneurons, perineuronal glia and axonal balloons in rats treated with iminodiproprionitrile
for 10 wk (282). This compound induces behavioral excitement and the marked hypertrophy of nerve cell bodies
and axons indicative of increased protein synthesis. While
the RNA content and base composition of neuron somas
and glial cells did not significantly differed from control
values, the RNA base composition of axonal balloons
became significantly different from that of neuronal cell
bodies. The RNA of axonal balloons had a consistently
lower adenine content (17.2 vs. 21.6%) but a higher content of cytosine (27.8 vs. 24.9%) and uracil (25.8 vs. 23.3%)
compared with the RNA of the neuron soma.
Using light and EM autoradiographic methods, axons
of the newt (Triturus viridescens) brachial nerves were
shown to incorporate [3H]uridine from 1 h to 8 days after
the intraperitoneal injection of this precursor (280). As
noted for the incorporation of the protein precursor
[3H]histidine in the same nerves (281), the decentralized
nerve manifested the same extent and pattern of RNA
precursor incorporation. As the silver grains were also
distributed over the nucleus and cytoplasm of Schwann
cells and over the myelin, these data supported the origin
of axonal RNA from periaxonal glia, in agreement with
similar data from other model systems (see sect. VIA).
In the peripheral nervous system of rats, the local
synthesis of axonal RNA (and protein) was demonstrated
by quantitative autoradiography of proximal stumps of
axotomized sciatic nerves in vivo (25). Following 30-min
incubation with radiolabeled precursors, newly synthesized RNA was present in Schwann cells, myelin sheaths,
and axons in order of decreasing concentrations. This
finding suggests that the periaxonal glial cell is the origin
of the axonal RNA in the sciatic nerve. Interestingly, the
peak of incorporation occurred 1 day after axotomy, in
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Evidence of the local synthesis of axonal RNA was
initially provided by microchemical analyses of axonal
and myelin RNA in transected proximal stumps of the
goldfish M-fiber (77). In specimens of 6 – 8 cm body length,
the RNA content in M-axons and myelin sheaths remained
essentially unchanged after axotomy. Nonetheless, their
base composition underwent remarkable changes as early
as 12 h after the lesion, with control values only reestablished after 20 –30 days. The main variation concerned the
A/G ratio that increased from 0.56 to 0.8 in the axon and
from 0.6 to 0.9 in the myelin sheath. These changes indicated rapid RNA turnover inside the axon and the myelin
sheath throughout the entire proximal segment of the
M-fiber. Their rapid onset and similar direction of base
composition changes suggested a common local origin of
the newly synthesized RNA, most probably in the oligodendroglial nucleus.
Additional evidence for a local synthesis of axonal
RNA was suggested by stimulation of the M-neuron
in vivo, accomplished by rotating caged goldfish around
their longitudinal axis at 60 cycles/min for 30 min (163).
These conditions increased the sensory input to the Maxon. The content of axonal RNA initially dropped 40% by
30 min after the end of the stimulation, but started to
increase in the following period. By 180 min, the content
of axonal RNA fully recovered. The RNA content in the
myelin sheath underwent similar changes, but with
marked variability. Nonetheless, the simultaneous increments in axonal and myelin RNA occurring between 30
and 180 min after the stimulation suggested that they
derived from the same source.
Incorporation of radiolabeled precursors into the
RNA of isolated M-fibers lacking their glial nuclei was
reported later (80). After incubation with radiolabeled
uridine and cytidine lasting to 21.5 h, newly synthesized
RNA was identified as 4S RNA by sedimentation on a
continuous sucrose density gradient. Despite the absence
of nuclei, the incorporation was strongly inhibited by
actinomycin D, a finding that suggested its mitochondrial
origin. Conversely, when the incorporation reaction was
allowed in the spinal cord segments in which M-fibers still
contain their glial cell bodies, newly synthesized RNA
isolated after a 7-h incubation period comprised 16S and
28 –30S rRNAs in addition to 4S RNA. Interestingly, most
of this newly synthesized RNA was in the axon (almost
80%). The synthesis of cytoplasmic rRNAs in the axons of
M-fibers containing glial nuclei, but not in M-fibers lacking
these nuclei, strongly support their transcription on the
oligodendrocyte nuclear DNA. The origin of 4S axonal
538
GIUDITTA ET AL.
VII. LOCAL SYNTHESIS OF PRESYNAPTIC RNA
A. Early Evidence
More than three decades ago, the local synthesis of a
presynaptic enzyme in a tissue lacking the cognate nerve
cell bodies suggested the local presence of the encoding
mRNA. In this early experiment, the activity of the heart
tyrosine hydroxylase (TH) was significantly increased a
few days after the administration of reserpine, which
depletes amine neurotransmitters from nerve terminals.
TH activity in the heart is due to the innervating cathecolaminergic nerve terminals. Interestingly, this effect was
markedly depressed by CXM, which was given when the
heart TH activity was on a sharp rise (295). The latter
observation and the substantial lack of involvement of
axonal transport suggested that presynaptic TH protein in
the heart may be locally synthesized and that the cathecolaminergic terminals contain TH mRNA.
Physiol Rev • VOL
More recently, RT-PCR analyses of the rat CNS extended this finding and demonstrated that TH mRNA is
present in the cathecholaminergic axons and nerve terminals of cerebellum, striatum, and pituitary intermediate
lobe. This conclusion is based on the absence of catecholaminergic cell bodies from these brain regions (228).
As observed for the heart enzyme, the content of TH
mRNA in the axons and nerve terminals may be locally
modulated. While unilateral damage to the cathecolaminergic pathway led to a significant decrease of the same
mRNA in the ispilateral striatum, reserpine induced a
marked increase of TH mRNA in the cerebellum. Hence,
the occurrence of protein synthesis and mRNA in the
nerve endings raised a fundamental question on the cellular origin of the components of protein synthesis machinery in the presynaptic domain.
B. Model Systems
1. Squid nerve endings
As mentioned in section IVA1, the unusually large
synaptosomes of squid optic lobes are exclusively generated by the nerve terminals of photoreceptor neurons.
They constitute the most abundant component of the
synaptosomal fraction and are essentially the only synaptosomes containing an active system of protein synthesis
(57, 58, 226). These unique properties and the remote
location of the cell bodies of these terminals suggested
that they could be profitably used to test the hypothesis of
the local origin of presynaptic RNA. Accordingly, the
large synaptosomes isolated from optic lobe slices that
had been incubated with [3H]uridine were expected to
contain radiolabeled RNA. If so, the result would have
proven the local transcription of presynaptic RNA, in view
of the anatomical absence of the parental photoreceptor
cell bodies from the incubated slices.
When the optic lobe slices were incubated for periods longer than 30 min, [3H]RNA was more abundant in
the synaptosomal fraction than in the microsomal/cytosolic fraction. However, its content in the synaptosomal
fraction more than doubled after 60 min. Hence, beyond
30 min, the newly synthesized RNA was largely prevalent in the synaptosomal fraction than in the microsomal/cytosolic compartment (92, 116). Such enrichment
was all the more remarkable in view of the abundance
of presynaptic nerve endings in squid optic lobe synaptosomes (Fig. 5B). The contribution of newly synthesized microsomal or cytosolic RNA was negligible. In
fact, when the incubated optic lobe slices containing
radiolabeled RNA were homogenized in a hyposmotic
buffer, the radioactive synaptosomal RNA was released
into the medium and nearly completely lost from the
synaptosomal fraction (92). The latter effect was expected from the swelling and bursting open of synap-
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good agreement with the period of maximal increment of
protein synthesis in transected hypoglossal nerves (298).
As also noted in the newt experiments (280), the intercellular transfer of RNA might have occurred in SchmidtLantermans incisures and/or in paranodal regions, i.e., in
loci where glial cytoplasm is abutting the axon (39, 192,
239).
Considerable support for the concept of intercellular
transfer of ribonucleoproteins from myelinated Schwann
cells to axons was obtained in peripheral nerves of the
mouse Wlds mutant in which the onset of Wallerian degeneration is markedly delayed (38, 123, 212). As shown
by conventional EM methods, in distal stumps of sciatic
nerves axotomized 1 wk earlier, the axons did not show
signs of degeneration but contained large numbers of
polysomes, some of which associated with membranes.
Polysomes appeared to have been synthesized locally by
surrounding glial cells, as they were associated with
newly synthesized RNA (F. Court and J. Alvarez, unpublished data). More recent studies with fluorescent antibody probes selectively identifying ribosomes, axoplasm,
and glial cytoplasm provided convincing evidence of a
transcellular transport of ribosomes from Schwann cells
to the axon (55). A comparable process also occurred in
the wild-type C57BL strain, albeit with lesser intensity.
With the use of conventional EM methods, double-walled
vesicles containing ribosome-like particles have been described at the internode axon-myelin interface of chicken
spinal motoneurons. Some of these vesicles are continuous with the subjacent axon. They are thought to be
involved in the transfer of glial ribosomes to the axon
(206). Comparable ultrastructural data have recently been
reported in the rat sciatic nerves examined with an antiribosomal polyclonal antibody (195).
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
C. Vertebrate Nerve Endings
1. Synaptosomal mitochondrial RNA
Since rat synaptosomal fractions contain mitochondria, notably at nerve terminals, it was of interest to
discern whether mitochondrial RNA synthesis occurred
in synaptosomal fractions. As shown by Attardi and collaborators (89), the synthesis of mitochondrial rRNA is
much more intense in synaptosomal preparations from
10-day-old rats than in rats of 30 days and is extremely
low in adult rats. In rats of 10 days, the majority of
radiolabeled RNAs included 16S, 12S, and 4S species.
Trace amounts of newly synthesized RNAs larger than 18S
were also observed. Interestingly, the synthesis of RNAs
larger than 4S was markedly inhibited by ethidium bromide (EB; a compound that specifically inhibits mitochondrial RNA synthesis). On the other hand, the inhibition of
4S RNA synthesis by EB was much weaker. Likewise,
RNAs larger than 4S satisfactorily annealed with mitochondrial DNA, but 4S RNA annealed less efficiently.
These data established the occurrence of mitochondrial
RNA synthesis in synaptosomal fractions, but implied that
synaptosomal 4S RNA may partly comprise nonmitochondrial species. Comparable data were reported with regard
to the synthesis of 4S RNA in the axons of isolated Mfibers (80). The latter findings were attributed to the
synthesis of mitochondrial RNA without direct evidence
(see sect. VIA2). Interestingly, EM autoradiographic data
on the rat synaptosomes showed that 50% of newly synthesized EB-sensitive RNA was present in presynaptic
synaptosomes.
FIG. 9. Presynaptic RNA of squid photoreceptor neurons is synthesized by local cells. A: colocalization of locally synthesized RNA (black dots)
and anti-syntaxin antibody (purple) in large synaptosomes. Scale bar, 10 ␮m. Synaptosomes were prepared from squid optic lobe slices incubated
2 h with [3H]uridine and processed for immunostaining using an antibody against squid syntaxin, a presynaptic marker protein. B: sedimentation
pattern of newly synthesized presynaptic RNA. Direction of sedimentation is to the right. Vertical arrows indicate the position of marker tRNA and
of the small and large subribosomal particles (s-RNA and l-RNA). Inset: percent distribution of radiolabeled RNA (F; mean ⫾ SE of 6 experiments)
and marker subribosomal particles (‚) in the gradient regions containing subribosomal particles. [Modified from Eyman et al. (92).]
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tosomes (110). Conclusive proof of the presence of
newly synthesized RNA in the large synaptosomes was
provided by autoradiographic analyses of the synaptosomal fraction. Essentially all radiolabeled RNA was
localized in the presynaptic synaptosomes identified by
their large size and their selective staining with a presynaptic antibody (Fig. 9A) (92, 116).
Similar to the experiments with the perfused squid
giant axon (see sect. VIA1), sedimentation analyses of the
locally synthesized presynaptic RNAs indicated that the
largest share of radiolabeled RNA was of relatively low
size, as it cosedimented with marker tRNA. In addition,
sizable amounts of RNA cosedimented with marker subribosomal particles, raising the possibility that the newly
synthesized RNA may include mRNA (Fig. 9B). The prevalence of low-molecular-weight RNAs at shorter incubation times had previously been noted for axoplasmic RNA
synthesized by isolated giant axons (229). On the whole,
axonal and presynaptic RNAs of low molecular weight
may undergo a remarkably high turnover. In principle,
they may be held responsible for the rapid modification of
the base composition of axonal RNA elicited by axotomy
(77), or for the rapid metabolic changes following stimulation (162). While the physiological significance of the
particular enrichment of small RNA species in axons and
nerve terminals is still largely unknown, some of these
small RNAs might be iRNAs involved in the modulation of
axonal and presynaptic protein synthesis. This interesting
possibility has been already observed and addressed in
neurons (189), dendrites (276), developing axons, and
growth cones (135), and more recently in axons of adult
mouse sciatic nerves (240).
539
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GIUDITTA ET AL.
2. Synaptosomal extramitochondrial RNA
VIII. TRANSFER OF GLIAL RNA TO THE
NEURON CELL BODY: THE
GLIA-NEURON UNIT
A. A Brief Overview
Except in very primitive organisms and some tissue
cultures, neuronal cell bodies, axons, nerve endings, and
dendrites are always in close contact with glial cells. In
oligodendrocytes and Schwann cells of myelinated axons,
most cytoplasm resides in the perinuclear region surrounding the myelin sheath. Glial cytoplasm is also present
Physiol Rev • VOL
10. Newly synthesized poly(A)⫹ RNA in rat brain synaptosomes has a short poly(A) tail. A: time course of the specific activity of
poly(A)⫹ RNA in the synaptosomal (‚) and the polysomal fractions (F)
of the rat brain after an intracranial injection of [3H]uridine. B: electrophoretic fractionation of the poly(A) segments isolated from synaptosomal (‚) and polysomal (F) poly(A)⫹ RNA prepared from rat brain 24 h
after an intracranial injection of [3H]uridine. Arrow, marker tRNA. Electrophoretic migration is to the right. Poly(A) segments were purified by
oligo(dT)cellulose chromatography from nuclease digests of radiolabeled poly(A)⫹ RNA. The major component of synaptosomal poly(A)
corresponded to 84 nt, while that of the polysomal fraction was twice as
large (169 nt). [Modified from DeLarco et al. (69).]
FIG.
in the spiral wrappings that abut the axon at the internodal
Schmidt-Lantermans incisures and paranodal regions (39,
192, 239). Even in nonmyelinated axons, glial cells form a
layer of variable thickness around axons. Morphological
evidence of the intimate relationship of glial cells with
neuronal cell bodies has been pointed out since the beginning of last century (140, 144). These observations
suggested the name of “trophospongium” for the system
of tiny channels presumed to mediate the exchange of
cytoplasmic organelles from glial cells to neurons. By the
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At variance with mitochondrial RNA synthesized in
presynaptic synaptosomes, the cellular origin of extramitochondrial presynaptic RNA has not been directly investigated in vertebrates. As a result, only some data may be
presumed to regard nerve terminals.
Synthesis of synaptosomal RNA was examined in
young rats after the intracranial injection of [3H]uridine
and [3H]adenosine (69). At the earliest time examined (1
h), the specific activity of synaptosomal RNA was approximately half that of mitochondrial RNA and one-fourth
that of polysomal RNA. Nonetheless, in the following time
period, the specific activity markedly increased in synaptosomes, but it increased to a lesser degree in mitochondria and polysomes. In addition, the specific activity of
synaptosomal poly(A)⫹ RNA remained higher than in
polysomes for several hours (Fig. 10A), indicating that the
turnover rate of poly(A)⫹ RNA was much higher in synaptosomes than in polysomes. Interestingly, the poly(A)
tail of newly synthesized synaptosomal poly(A)⫹ RNA
was only half as long as that in polysomes (Fig. 10B). This
feature suggests that most newly synthesized synaptosomal poly(A)⫹ RNA may have a presynaptic localization.
Indeed, the poly(A) tail of axonal mRNAs in the mammalian hypothalamic-hypophysial tract is markedly shorter
than that of perikaryal mRNAs (232, 233). As polyadenylation of mRNA is implicated in the regulation of the
message (218, 269), this interesting feature of newly synthesized synaptosomal poly(A)⫹ RNA may provide an
important clue as to its role.
The subcellular distribution of newly synthesized
brain RNA was also determined in adult rats 1 h after the
intraventricular injection of [3H]uridine (61). At this time,
synaptosomes contained a lesser amount of newly synthesized RNA than mitochondria and microsomes. However, the proportion of their radiolabeled poly(A)⫹ RNA
was somewhat higher than in mitochondria and markedly
higher than in microsomes and nuclei. These data confirmed the surprisingly high content and turnover of
newly synthesized poly(A)⫹ RNA in rat synaptosomes.
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
B. Experiments With Single Cells
Microchemical methods of analyzing microdissected
tissue samples (82) provided the first data on the biochemical composition of glial and nerve cells, notably in
the experimental conditions of enhanced nervous activity
or learning. An important issue raised in this field concerned the degree of mutual contamination of the glial
and nerve cell samples and the contamination by extraneous structures (193). The purity of microdissected glial
cells and neuron cell bodies has been largely assessed by
visual inspection, and generally reported to be as high as
85–90%. On a first approximation, isolated neuronal nuclei
Physiol Rev • VOL
and cell bodies, especially of large size, have been considered least contaminated, while clumps of glial cells are
likely to be less pure.
1. Enhanced nervous activity
The identification of morphological changes induced
by exposure of selected groups of nerve cells to functional loads (or to axotomy) was one of the main issues
pursued in the later part of the 19th century, notably after
the identification of Nissl bodies in neuronal perikarya.
These studies were greatly stimulated by the development
of quantitative cytospectrophotometric methods to determine nucleic acids in single cells (for a rigorous example,
see Ref. 11; for a review, see Ref. 45). An even stronger
impetus was provided by the later availability of micromanipulators and microscale biochemical methods with
which the RNA content and base composition of microdissected cell samples could be measured at the picogram
level (82). The RNA base composition was determined
with the standard errors of the mean typically within 1–2%
of the average (84, 151). It was through this method that
the existence of mRNA in single eukaryotic cells (initially
termed “nonribosomal DNA-like RNA”) was first distinguished from rRNA or tRNA. A high value of the GC/AU
ratio indicated the prevalence of rRNA, while lower values suggested the presence of mRNA.
In the early neurobiological investigations of this
kind, the lateral vestibular nucleus of Deiters became an
experimental system of choice, as its unusually large neurons could be hand-dissected with relative ease (Fig. 11). In
rabbits, an average Deiters neuron was reported to have a
volume of 93,000 ␮m3 and a surface area of 50,000 ␮m2,
most of which belonged to dendrites (60%) (158). Stained
with a dilute solution of methylene blue, each neuron
appeared to still harbor ⬃10,000 nerve terminals. The dry
weights of its cytoplasm, nucleus, and nucleolus corresponded to 20,094, 800, and 100 pg, respectively. Each
Deiters neuron was surrounded by 35– 40 glial cells,
mostly oligodendrocytes. Because of their distinct size,
Deiters neurons could be analyzed singly, but the glial
cells needed to be grouped in clumps of six to eight cells
trimmed to about the same volume of the nerve cell. This
constraint is likely to account for the relatively higher
degree of contamination of the glial samples.
Although electrophysiological activity of Deiters neurons was never recorded, they were assumed to be stimulated through the vestibular input when rabbits were
rotated 120° horizontally and 30° vertically at 30 rpm
(with their head away from the axis of rotation). This
treatment was routinely imposed 25 min each day for 7
consecutive days. At the end of this period, the dry weight
of the neuron increased ⬃12%, mostly through an increment in density. A comparable increase also occurred
after 1 day of stimulation. The content of protein in-
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same token, glial cells were once named “trophocytes.”
Comparable observations with better analytical methods
were later made in a large number of vertebrate and
invertebrate nervous systems, often leading to suggestions of underlying exchanges of macromolecules between glia and neurons (for reviews, see Refs. 68, 193,
230, 243, 292, 311, 320, 327). Glial cells have also been
shown 1) to display resting membrane potentials of medium or high values that are liable to undergo slow and
relatively long-lasting modifications (102, 193, 194, 312)
and 2) to be endowed with a host of ion channels and
membrane receptors for many types of neurotransmitters
(20, 207, 241, 270).
These basic features suggest that glia and neurons
are involved in mutually cooperative roles. In view of the
widespread intercellular cooperation present in multicellular organisms, interactions of glial and nerve cells may
be considered a more complex and specialized example
of a general phenomenon. Cell-to-cell interactions are
usually selective and dynamic, and the involved partner
cells are likely to inform each other of their functional
states before engaging in coordinated actions. Experimental evidence supporting intense exchange of signals
among glial cells and neuronal domains has substantially
increased in recent years, largely with regard to the mammalian perisynaptic glia (12, 28, 272, 314). Cooperation
between glial cells and neurons is likely to have started in
the early stages of phylogenetic evolution of the nervous
system, presumably to maintain a proper ionic milieu in
the extracellular space or to perform basic collaborative
functions. In time, with the progressive increase in size
and complexity of the nervous system in metazoan animals, the glia-neuron cooperation attained a wider scope
and gave rise to functional specializations allowing more
complex performances. An obvious example is the extensive myelin wrappings around vertebrate axons.
For a detailed basic knowledge on the classification,
embryologic origin, and properties of glial cells, the readers are referred to the informative publications covering
this field (19, 68, 193, 311, 320, 327).
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GIUDITTA ET AL.
creased from 16,080 to 18,060 pg/cell, and that of RNA
from 1,545 to 1,612 pg/cell. In contrast to these effects, the
content of RNA in the same volume of glial cells underwent a marked decrease (from 123 to 85 pg). Comparable
inverse changes in glia and nerve cells were also observed
with regard to cytochrome oxidase, while succinoxidase
increased in neurons (2.7-fold) but remained essentially
unchanged in the glial sample. Under resting conditions,
the activity of both enzymes was consistently higher in
the glial sample. These data were interpreted to reflect a
functional cooperation between neurons and glia (the
neuron-glia supracellular unit) that was poised to give
neurons “metabolic priority” in oxidative metabolism and
to provide neurons with key glial components such as
RNA under conditions of enhanced neuronal activity. Anaerobic glycolysis also showed inverse changes in neurons and glial cells, but in the opposite direction (130).
Under a chronic work load, neurons enhanced their oxiPhysiol Rev • VOL
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FIG. 11. Single neurons dissected from the brain for microchemical
analyses. A: micrograph of a large Deiters neuron obtained from an adult
rabbit by freehand dissection. The neuron cell body and basal dendrites
are densely covered with synaptic knobs. B: micrograph of a Deiters
neuron whose synaptic knobs have been detached from the cell body
and basal dendrites by a 30-min incubation in isotonic medium. The RNA
content and base composition of single Deiters neurons were determined under conditions of enhanced vestibular activity or following
training. [Modified from Hydén et al. (159).]
dative metabolism and reduced anaerobic glycolysis,
while glial cells did the opposite. These responses were
specific for perineuronal glia, as pericapillary glia was
only barely affected (129). Hence, the physiological significance of this metabolic shift in glia was probably
related to the metabolic need of adjacent neurons.
Comparable changes were induced by short-lasting
treatments of live rabbits or rats with pharmacological
compounds. In rabbits, administration of the malononitrile dimer (TRIAP: 1,1,3-tricyano-2-amino-1-propene) for
1 h elicited marked inverse changes in protein and RNA of
Deiters neurons and glia (86). The neuronal RNA and
protein increased by nearly 25%, while the glial RNA
decreased by 45%. In addition, TRIAP modified the base
composition of neuronal RNA by increasing G and decreasing C. Conversely, in glial cells close to the neuronal
perikaryon, the RNA base composition changed in the
opposite direction, in that G markedly decreased and C
markedly increased. Comparable data were later reported
in Deiters neurons and glia of rabbits treated for 1 h with
tranylcyclopropylamine, an inhibitor of monoamino oxidase (155).
The Deiters neurons also provided insight into the
intracellular transfer of RNA. In fact, when rabbits were
treated with TRIAP for 1 h, the RNA content of the Deiters
cell body increased (see above), but the RNA content of
the cell nucleus decreased 16%. The nuclear RNA also
underwent base composition changes, with a marked decrease in uracil (151). Hence, in addition to the RNA
transfer from glia, the neuronal cytoplasm received a
U-rich RNA fraction synthesized in the nucleus. These
data may hardly be attributed to contamination by extraneous material, as assumed for the glial changes (193). In
addition, they imply that some neuronal RNA responses
may be attributed to the synthetic capacity of the neuron.
When the RNA changes induced by TRIAP were calculated with regard to the fraction of RNA lost or gained
by neurons or glia, striking similarities emerged (156,
157). The RNA gained by neurons had a base content of
22.5% A, 37.7% G, 21% C, and 18.8% U, which was strikingly similar to the base composition of the RNA lost by
the glial sample (21.2% A, 37.8% G, 23% C, 17.6% U).
Furthermore, the RNA gained by each neuron (570 pg)
approximately corresponded to the RNA lost by its surrounding glia. These similarities were interpreted to support the hypothesis of an intercellular transfer of RNA. At
variance with these data, chronic administration of TRIAP
to mice and rats for 2–5 wk did not induce RNA changes
in neurons and glial cells, although the treatment elicited
marked increments in neuron size and neurofilament content, and in myelin thinning (282). These results supported the view that significant changes in the RNA of the
neuron cell body and surrounding glia only occurred after
acute stimulation, but were overshadowed by hypertrophic processes elicited by chronic stimulation (318 –320).
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
Physiol Rev • VOL
losses occurred in neuronal and glial RNAs (⫺26 and
⫺19%, respectively). In addition, the RNA base composition of both neuron and glia showed marked parallel
changes (⫺2.7% A, and ⫹2.2% C), suggesting the loss of a
short-lived RNA fraction rich in A and poor in C. On the
other hand, when preparations incubated with actinomycin D were continuously stimulated for 8 h, the loss of
neuronal RNA was conspicuously alleviated (⫺10% instead of ⫺26%), but the loss of glial RNA remained high
(⫺15% instead of ⫺19%). In addition, when a control
preparation not inhibited by actinomycin D was continuously stimulated for 8 h, the RNA content of the surrounding glia significantly decreased (⫺21%) while the RNA
content and base composition of the stretch receptor
neuron did not significantly change. These results imply
that the stimulated neuron is able to maintain its RNA
content through a route other than RNA synthesis, presumably through the uptake of glial RNA.
2. Learning
More intriguing results were reported with regard to
the changes in the content and base composition of neuronal and glial RNA under learning conditions (152, 153).
In the first experiment (152), adult rats were trained to
climb a long wire placed at 45° from the cage floor to
reach the food provided on a high platform. Rats trained
in daily sessions of 45 min needed 4 –5 days to perform a
full walk. Thereafter, the number of daily visits to the
platform increased from 3– 4 to ⬃20 in the following 3
days. When food was removed from the platform, the
frequency of trips started to decrease. In this experimental protocol (balancing task), the total content of cellular
RNA in Deiters neurons slightly increased 8 days after the
beginning of training, with no change in its base composition. However, the A/U ratio of the nuclear RNA increased from 1.06 to 1.35. Likewise, a marked increment
in A and a decrease in C occurred in glial RNA. These
changes suggested the prevalent synthesis of DNA-like
RNA (mRNA) during the learning process. This shift in
RNA base composition was specific to learning, as control
rats subjected to vestibular stimulation did not display
changes in A or U contents. The RNA change in the
nucleus of experienced rats was not persistent, as the A
and U contents returned to the normal level when rats
were not trained for a day.
Comparable results were reported in cortical neurons of rats trained in a reversed handedness protocol
(154). In this experiment, rats were initially selected according to the preferred paw they used to gather food
from a glass cylinder placed 5 cm above the cage floor.
Right-handed rats were then forced to use the left paw by
an obstacle. Training sessions of 25 min were scheduled
twice a day. After 200 forced reaches, memory was retained for at least 9 mo. Four days after the start of
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RNA responses to enhanced neuronal activity were
also reported in neurons and glia cells examined by ultraviolet cytospectrophotometric methods (260). One advantage of these methods is that the measurement is made in
situ, without microdissection of individual cells. In anesthetized cats, high-frequency stimulation of the preganglionic nerve to the superior cervical ganglia for 3 h induced
a significant increase of RNA in neuronal cytoplasm
(44%), but a 30% decrease of glial OD265, an index of its
nucleic acid content. Since the cellular DNA content is
not expected to change, these values were attributed to
variations of the RNA content. The inverse pattern of
changes in neurons and glia was similar to that obtained
using microchemical methods (see above). Hence, this
weakened the possibility that the comparable changes
detected with the microchemical methods were due to the
dendritic RNA entangled in the glial sample (see also sect.
VIIIB3). Comparable results were later obtained with different neurons and their corresponding glial cells analyzed with the same method (261). In agreement with
previous data, these results suggested that an increment
in neuronal RNA and protein was associated with the loss
of glial RNA only if the stimulation was sufficiently intense and/or prolonged. Under conditions of greater functional load, neuronal and glial RNAs could both decrease.
Conversely, in the following rest period, RNA recovery
first occurred in glial cells and then, with delay, in neurons. Hence, glial cells synthesize RNA at a faster rate
than neurons, in agreement with other experiments including direct analyses of microdissected hypoglossal
neurons and glial cells (63).
The slowly adapting stretch receptor neurons of the
lobster (Homarus grammarus) provided a considerable
advantage as an experimental model system, as they allowed both microchemical RNA analyses and electrophysiological monitoring of neuronal activity. In one
study, this neuron was stimulated up to 6 h by stretching
the muscle. This created an average frequency of action
potentials close to 5/s and a total number of spikes ranging from 24,000 to 132,000. Compared with the control
cells, the RNA content was not modified by activity, but
significant increments occurred in its A content, A/U ratio,
and purine-to-pyrimidine ratio (126). This experiment is
remarkable as it confirmed that the base composition of
neuronal RNA could be significantly modified by a physiological work load imposed under rigorous experimental
conditions. In the follow-up investigation, the same neuronal preparation was used to examine the effect of more
intense stimulations (average spike frequency of 5–10/s)
that lasted longer (up to 24 h) and yielded a higher total
number of spikes (from 175,000 to 600,000) (85). The high
turnover of neuronal and glial RNA was demonstrated by
incubating for 8 h a nonstimulated preparation with the
transcription inhibitor actinomycin D. Under these conditions of complete inhibition of RNA synthesis, marked
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3. Commentary
The interpretation of these microchemical data has
been the target of a pointed criticism regarding technical
and theoretical issues. Notably, in an alternative interpretation (193), the inverse changes reported in neurons and
glia were attributed to the intracellular shift of RNA moving to the neuron soma from dendrites entrapped in the
glial samples. Additional criticism regarded the general
absence of electrophysiological monitoring of neuronal
activity and the arbitrary selection of the analyzed cells.
Forty years after such a negative assessment of this entire
field, the growing body of evidence that the axon domain
contains local systems of protein synthesis fed by glial
transcripts calls for the reconsideration of those pioneering data. Indeed, their consistency and specificity support
the view that those early studies opened more encompassing and intriguing perspectives in the understanding
of the cellular biology of the nervous system.
True, most experiments on the effects of increased
functional loads did not include monitoring by electrophysiological methods, but this was actually done in a few
experiments (see Refs. 85, 126, 260, 261). True, it is surprising that randomly collected neurons of the same type
displayed consistently similar biochemical changes when
exposed to a certain experimental condition. Nonetheless, objections to an imperfect experimental design do
not imply that neural activity did not increase, nor that a
given type of neuron cannot respond in a similar fashion
to a given challenge. These considerations convey doubts,
not disproof, and doubts in science should lead to better
designed experiments and appropriate controls, rather
than to the neglect of a whole field of endeavor.
Furthermore, the alternative suggestion that the inverse changes detected in glial and nerve cells may reflect
an intracellular translocation of RNA rather than an interPhysiol Rev • VOL
cellular exchange is still lacking experimental support.
Dendrites are known to import RNA from the neuron cell
body rather than exporting it to the perikaryon, or even
less likely to the neuronal nucleus. This early criticism
appears to have been largely based on the lack of information on intercellular exchanges of materials, which is
now well known to occur in eukaryotic cells, such as
follicular cells and oocytes (217, 238). The data supporting intercellular transport of glial RNA to the neuron soma
have also been corroborated by cytospectrophotometric
methods that may hardly be blamed for confusing a glial
cell with a dendrite (260, 261). As reviewed in this article,
intercellular transport of newly synthesized RNA from
periaxonal glia to the axon has been demonstrated in
vertebrate (25, 80, 177, 280) and invertebrate species (9,
62, 92, 117, 229, 265), as well as between perisynaptic cells
and squid nerve terminals (92, 116).
While the latter data make it more likely that comparable macromolecular exchanges may occur between
perisomatic glia and neuron cell bodies, it is to be noted
that the neuronal response to a chronic functional load
consists in a long-lasting wave of enhanced RNA synthesis, as extensively documented by increments in the cytoplasm-to-nuclear ratio of neuronal radiolabeled RNA
(318 –320). Hence, at least some of the changes in neuronal RNA are due to the transcriptional capacity of neurons. These two modes of providing somatic RNA are not
mutually exclusive. In neurons with a relatively limited
cytoplasmic mass, deriving RNA from the cell nucleus
may be sufficient, especially under conditions of moderate activity. On the other hand, neurons with an overwhelming cytoplasmic mass, notably if engaged in intense
activity, might also require the additional supply of glial
RNA (see sect. VI).
Such a glial assistance is also suggested by the increased number of perineuronal glial cells concomitant
with the progressive increment in the mass of neuronal
cytoplasm. A clear example is provided by the striking
linear correlation between the number of oligodendrocytes surrounding the neuron cell bodies of Clark’s column in the spinal cord and the length of their axons (100,
267). This correlation remains valid whether the axon
length varies with the segmental position of its soma
along the spinal cord or according to the body size of
mammalian species (from mouse to whale). More recently, the number of perisomatic glial cells in spinal
ganglia was also reported to increase with the volume of
the neuron cell body (203, 227). Since cell body volume is
related to axon length, the former variable may be more
directly conditioning the ratio of glial number to axon
length. Such a tightly paired association of neuron size
with perineuronal glial number suggests that one of the
major factors of this striking outcome regards the multiplication of glial genomes. A comparable multiplication at
a genomic scale occurs in the follicular cells of developing
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training, analyses were made on groups of 10 pyramidal
neurons of layers 5 and 6 that were shown to be required
for learning the task. This procedure was followed because each nerve cell only contained 22 pg RNA. When
cortical neurons of the right hemisphere were compared
with the homologous neurons of the left hemisphere (assumed not to be involved in learning this task), their RNA
content was ⬃23% higher and its base composition presented a marked decrease in C and small increments in A,
G, and U. The lowered GC/AU ratio (from 1.72 to 1.51)
suggested a significant increment of mRNA. This effect
was also specific to learning, as in the experiments with
control rats the RNA of pyramidal neurons from either
cortical side was identical in content and base composition. The influence of contingent factors was excluded, as
when rats used their preferred right paw, neurons of the
left cortex only showed a minor increase in RNA and no
change in its base composition.
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
IX. THE LOCAL SYSTEM OF GENE
EXPRESSION IN AXONS AND
NERVE TERMINALS
The concept of a supracellular “glia-neuron unit” was
proposed ante litteram at the beginning of last century
(144), but gained momentum only thanks to the extensive
microanalytical work on the transfer of RNA and protein
from glia to the neuron cell body (for a review, see Ref.
157). While the latter data were severely criticized (193),
and the possibility of glia-to-neuron transfer of RNA was
largely forgotten, the concept of the glia-neuron unit was
revived in studies of the local synthesis of proteins in
isolated giant axons of crayfish (30, 147) and squid (104,
200, 201, 277, 303). The squid data were taken to support
the idea that locally synthesized axonal proteins exclusively derive from periaxonal glia, although later experiments demonstrated this view to be incorrect (see sects.
III and IV).
The demonstration that axons and nerve terminals
possess autonomous systems of protein synthesis does
not exclude the possibility that glial proteins may be
transferred to these peripheral neuronal domains. Indeed,
Physiol Rev • VOL
a number of experimental data demonstrate the transfer
of glial proteins (and RNA) to the neuron cell body (see
sect. VIII), to the squid giant axon (sect. VIA1) and nerve
endings (sect. VII), and to the mouse Wlds axons (see sect.
VIB). Furthermore, axons, nerve terminals, and neuron
cell bodies are capable of protein uptake (115, 145, 188,
191, 321). Hence, in each neuronal domain, it is reasonable to ask what proteins are locally synthesized and what
others are derived from glia. Of the locally synthesized
proteins, only some have been identified, but most of
them are unknown and of doubtful origin. As exemplified
in Figure 12 for the squid giant axon, the population of
proteins synthesized by axoplasm may be quite different
from those transferred from glia. In future studies, the
comparison between locally synthesized and glia-transferred proteins may reveal interesting correlations, especially if basal data will be compared with those gathered
under conditions of enhanced activity.
As outlined in the previous sections, the neuron soma
cannot be considered the only source of axonal and presynaptic proteins. Rather, proteins of the axon domain
also derive from local systems of gene expression that
integrate glial transcription events with the translation
machinery operating in axons and presynaptic terminals.
These extrasomatic systems provide the neuronal periphery with the trophic support needed to implement responses that are largely independent from the neuron cell
body. These local systems of gene expression are likely to
be the phylogenetic consequence of the massive cytoplasmic growth of the axon domain that came to outpace the
trophic capacities of the neuron soma (315). Clearly, this
novel concept may only be accepted on the basis of the
following evidence: 1) that glial cells transcribe RNA and
deliver it to axons and nerve endings, 2) that glial transcripts are translated by the cytoplasmic systems of protein synthesis present in axons and presynaptic sites, and
3) that the latter systems are modulated by local transcription processes. The aforementioned conditions are
all supported by the experimental evidence in axons,
albeit to a different degree in different axons. On the
other hand, some evidence is lacking for nerve terminals,
notably for mammalian presynaptic endings, chiefly because of the lack of suitably designed experiments. Accordingly, the supportive data will be summarized separately for the two domains. Such a distinction is also
justified by their different functional roles. The missing
elements from this novel mosaic will be highlighted as
they occur.
A. Axons
The presence of a cytoplasmic system of protein
synthesis has been convincingly documented in all axons
that have been investigated. The main features of this
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oocytes (217, 238). It may be noted that the role played by
multiple sets of cell genomes may not be substantially
dissimilar from the amplification of more restricted DNA
regions, except for the global extent of its trophic potential.
It may nonetheless be asked whether the RNA lost
from perineuronal glia is exclusively targeted to the neuron cell body. An intriguing possibility is suggested by the
alleged presence of a very large number of nerve terminals attached to the surface of dissected neuronal
perikarya. In Deiters neurons, this number is estimated to
be 10,000 (Fig. 11) (158). Hence, the increase in the cell
body RNA could be attributed to its associated nerve
endings, in analogy to the transfer of newly synthesized
RNA from perisynaptic cells to squid presynaptic terminals (92, 116). According to this hypothesis, glial RNA
would be transferred to a neuron domain incapable of
transcribing but capable of translating cytoplasmic RNAs.
A quick supply of transcripts to this domain would allow
its quick response to rapidly changing cell commitments.
Whatever the ultimate target of perisomatic glial RNA
may be, it is worth noting that the rate of RNA synthesis
in glial cells is at least twice as high as in neurons when
calculated per unit amount of RNA (63). This observation
implies that the turnover rate of glial RNA is twice as high
as in neurons. Although the content of RNA in glial cells
is generally much lower than in neurons, the large number
of glial cells per neuron and the high turnover of glial RNA
are likely to fulfill a role presumably related to its fleeting
export function.
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GIUDITTA ET AL.
B. Nerve Terminals
FIG. 12. SDS-PAGE patterns of newly synthesized axonal proteins
from squid giant axons. A: radiolabeled proteins collected from an
internally perfused giant axon. B: radiolabeled proteins from the extruded axoplasm of the contralateral axon. Both axons were incubated
2 h with [35S]methionine. The proteins shown in A are assumed to derive
largely from the periaxonal glia, as most axoplasm was extruded from
the axon before starting the perfusion. On the other hand, to obtain the
proteins shown in B, the contralateral axon was incubated with the
radiolabeled amino acid, and the axoplasm was extruded at the end of
the incubation. Under this condition, most proteins are assumed to be
synthesized by the axonal system of protein synthesis. (From M. Eyman
and A. Giuditta, unpublished data.)
system have been well characterized in model preparations of vertebrate and invertebrate species in terms of
macromolecular components and translation products
(see sect. III). Cogent data are likewise available with
regard to the local synthesis of axonal RNAs. This information was initially obtained from axonal model systems
of particularly large size that are more readily amenable
to suitable analyses. Nonetheless, a number of other axons of normal size have provided supporting data from
Physiol Rev • VOL
Presynaptic protein synthesis has been convincingly
demonstrated and characterized in a squid model preparation (see sect. IVA1). As to mammalian nerve endings, a
wealth of data supporting their contribution to synaptosomal protein synthesis has been presented and critically
discussed in section IVB. Direct evidence is provided by
EM autoradiographic data demonstrating that at least half
of the newly synthesized synaptosomal proteins are localized in presynaptic synaptosomes (54, 105). A more detailed survey of the available evidence is reported in a
previous review article (7).
The existence of cytoplasmic protein synthesis in
mammalian nerve endings is also indirectly suggested by
the existence of a translation system in growth cones (66,
190). In a number of experiments, this system has been
shown to play an essential role in axon guidance during
development (37, 42, 204, 231, 332, 333). Hence, the local
system of protein synthesis is highly functional. The dramatic burst of protein synthesis that occurs at the very
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investigations with autoradiographic or microchemical
methods (see sect. VI). In the isolated Mauthner fiber of
the goldfish, the synthesis of axonal rRNAs only occurs in
the presence of the glial nucleus. In the isolated squid
giant axon, locally synthesized axonal RNAs have been
identified as cytoplasmic tRNA, rRNAs, and poly(A)⫹ RNA,
which require nuclear DNA for transcription. Hence, the
data imply transcription processes occurring in periaxonal glia, the only nucleus-containing cell element present
in the isolated axon. Direct evidence of their glial origin
has been recently provided by the involvement of glial
glutamatergic and cholinergic receptors in the delivery of
newly synthesized RNA to the perfusate of giant axons.
In mammalian peripheral nerves, the identification of
the mRNAs coding for neuron-specific NF proteins in
Schwann cells (95, 170, 271, 286) is consistent with a glial
transcription of axonal mRNAs, and with their transcellular transfer to the axon. More recently, the latter process has been elegantly demonstrated in the myelinated
axons of mouse Wlds mutants (55). In addition, axonal
protein synthesis has been shown to depend on local
transcription events. The first convincing observations of
this modulatory process were made more than 30 years
ago with regard to the resynthesis of AChE in intact and
axotomized mammalian nerves. In these experiments, the
local synthesis of axonal proteins was shown to be markedly inhibited by RNase or by inhibitors of RNA synthesis
such as 5-fluororotate, and markedly stimulated by a paradoxical effect of actinomycin D (see sect. III). Hence,
local protein synthesis in vertebrate axons is also largely
dependent on the transcriptional contribution of periaxonal glial cells.
LOCAL GENE EXPRESSION IN THE AXON DOMAIN
X. CONCLUSION AND NOVEL PERSPECTIVES
The original supracellular concept of the “glia-neuron
unit” takes on a deeper and more stimulating meaning in
the present proposal, as it leads to the view that the axon
domain is served by a distinct local gene expression system based on the transcellular delivery of glial transcripts.
This feature allows peripheral regions of the neuron to
promptly respond to changing local challenges, thereby
Physiol Rev • VOL
bypassing the slower communications with the cell body.
Such a mechanism is of special euristic value for the
nerve endings, as it also allows their highly selective
plastic modifications (128, 220). From this point of view,
a local gene expression system may also be assumed to
exist in dendrites, notably those displaying an extensive
arborization (e.g., Purkinje or pyramidal neurons). Nonetheless, macromolecular contributions from the neuron
cell body are expected in all peripheral neuronal domains.
A schematic representation of the view purporting the cell
body origin of all neuronal RNAs is shown in Figure 13A.
Conversely, the view proposed and discussed in the
present article is presented in Figure 13B. The distinctive
roles of the somatic and glial source of neuronal RNAs
remain to be determined under basal conditions and during enhanced physiological activity.
As implied by our considerations, the main consequence of the novel version of the glia-neuron hypothesis
regards the molecular basis of the substantial autonomy
that axons and nerve endings implement in their plastic
responses. Such autonomy is manifested in both physiological and pathological contexts. Several examples have
been provided with regard to the long-term structural and
functional changes of differentiated axons (for a review,
see Ref. 7) and nerve terminals (94, 128, 168, 219). These
changes require the activation of gene expression systems
FIG. 13. Schematic representation of the cellular origin of RNA in
well-differentiated neurons. A: the widely held concept that somatic,
axonal, and presynaptic RNAs exclusively derive from the neuronal
nucleus, as indicated by the blue lines ending with arrows. To avoid
unnecessary crowding of the panel, the lines indicating the obvious
nuclear origin of cytoplasmic glial RNA have been omitted. B: the view
discussed in this article that axonal and presynaptic RNAs may derive
from periaxonal and perisynaptic glial cells, as indicated by the red lines
ending with arrows. The dotted blue line originating in the neuronal
nucleus is a reminder of the still putative somatic derivation of axonal
and presynaptic RNAs. In addition, the dotted red line going from the
perisomatic glia to the neuron soma indicates that this transcellular
route may only operate under conditions of enhanced physiological
activity.
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end of the proximal stump of axotomized neurons (174,
177) demonstrates the participation of local protein synthesis in axon regeneration. Its physiological significance
has been recently elucidated by investigations of regenerating axons in various model systems (75, 101, 133, 134,
146, 215, 254, 255, 302, 330, 339; for reviews, see Refs. 23,
331). There is hardly any reason to assume that growth
cones would silence their protein synthesis soon after
reaching their targets and maturing into nerve endings. In
mature neurons, nerve endings are farthest away from the
nerve cell body, and their proteins undergo continuous
turnover to comply with rapid changes in synaptic plasticity. Experimental data demonstrate that the physiological need to replenish the constituent proteins is met by
local protein synthesis (see sect. IVA2).
Direct evidence for local synthesis of presynaptic
RNA has been recently documented in the nerve terminals
of squid photoreceptor neurons (92, 116). To our knowledge, comparable evidence is not yet available for the
mammalian nerve endings, chiefly because of the lack of
suitably designed experiments. Nonetheless, the following data provide some initial encouraging support: 1) the
local modulation of TH mRNA in brain regions containing
cathecolaminergic nerve endings but not their cell bodies
(228), and 2) the prevalence of poly(A)⫹ RNA and of
newly synthesized poly(A)⫹ RNA in rat synaptosomal
fractions (59 – 61, 69). The major presynaptic localization
of this RNA is suggested by the markedly shorter length of
its poly(A) tail compared with microsomal poly(A)⫹ RNA
derived from cell bodies (Fig. 10B). A shorter poly(A) tail
is a characteristic feature of axonal mRNAs (232).
A key link requiring experimental verification concerns the local transcriptional modulation of presynaptic
protein synthesis. This may be considered the main missing tessera of the proposed novel mosaic. As the local
transcription of presynaptic RNA has been reported only
recently with regard to a squid model system (92, 116), it
is not surprising that this tessera is still missing. In some
respects, and with a due sense of proportions, the present
state of the glia-neuron hypothesis may be reminiscent of
the Metchinikoff’s map of chemical elements at the time
of its compilation. Of course, we have good reasons to
believe that even the presently missing links will be
brought to light in the near future.
547
548
GIUDITTA ET AL.
ACKNOWLEDGMENTS
This paper is in honor of the insight and endurance of Dr.
Edward Koenig whose pioneering investigations have greatly
contributed to our present understanding of this basic neurobiological field.
We gratefully acknowledge the helpful comments and suggestions made on the manuscript by our colleagues Drs. J.
Alvarez, B. B. Kaplan, E. Koenig, and R. Martin.
Squid data have largely been obtained at the Marine Biological Laboratory (Woods Hole, MA) and, in part, at the “Anton
Dohrn” Zoological Station (Naples, Italy).
We deeply apologize for the possible involuntary omission
of some key references.
Address for reprint requests and other correspondence: A.
Giuditta, Dipartimento delle Scienze Biologiche, Via Mezzocannone 8, Napoli 80134, Italy (e-mail: [email protected]).
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