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The Journal of Neuroscience, June 15, 2002, 22(12):4955-4963
Axonally Transported Peripheral Signals Regulate  -Internexin
Expression in Regenerating Motoneurons
Tanya S.
McGraw1,
J.
Parker
Mickle2,
Gerry
Shaw1, and
Wolfgang J.
Streit1
Departments of 1 Neuroscience and
2 Neurological Surgery, University of Florida College of
Medicine and McKnight Brain Institute, Gainesville, Florida 32611
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ABSTRACT |
The class IV neuronal intermediate filament (IF) family
proteins includes the neurofilament (NF) triplet proteins NF-L, NF-M, and NF-H and also the more recently characterized  -internexin-NF66. It is well established that NF-L, -M, and -H protein and mRNA are
downregulated after peripheral nerve injury. We examined -internexin protein expression after three facial nerve lesion paradigms: crush,
transection, and resection. -Internexin immunoreactivity was absent
in the perikarya of uninjured facial motoneurons but increased
dramatically in all three injury paradigms, with maximum immunoreactivity observed at 7 d after injury. Twenty-eight days after nerve crush or transection, there was a dramatic decrease in the
number of -internexin-positive cells. In contrast, -internexin remained elevated 28 d after nerve resection, an injury that
hinders regeneration and target reinnervation. In situ
hybridization studies showed an increase in -internexin mRNA
expression in the facial nucleus at 7 and 14 d after injury.
Retrograde transport of fluorogold from the whisker pads to the
facial nucleus was seen only in motoneurons that lacked
-internexin immunoreactivity, supporting the idea that target
reinnervation and inhibitory signals from the periphery regulate
the expression of -internexin. Blockage of axonal transport through
local colchicine application induced strong immunoreactivity in
motoneurons. -Internexin expression was also examined after central
axotomy of rubrospinal neurons, which constitutively show -internexin immunoreactivity. After rubrospinal tractotomy,
-internexin immunoreactivity transiently increased by 7 d after
injury but returned to control levels by 14 d. We conclude that
-internexin upregulation in injured motoneurons suggests a role for
this IF protein in neuronal regeneration.
Key words:
axotomy; regeneration; axonal transport; neurofilament
proteins; motoneurons; -internexin
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INTRODUCTION |
-Internexin, also known as NF66,
is a neurofilament (NF) subunit protein distinct from the 68 kDa NF-L
protein, which has an apparent SDS-PAGE molecular weight of 64-66 kDa.
It was originally named -internexin because of an apparent
ability to bind intermediate filaments together (Pachter and Liem,
1985 ), and, later, on determination of the primary amino acid sequence,
it was recognized to be a bona fide member of the intermediate or 10 nm
filament protein family (Fliegner et al., 1990). The same protein was
independently discovered as a neuronal intermediate filament (IF)
subunit by another group, who named it NF66 (Chiu et al., 1989). Based
on amino acid sequence homology of the protein and the intronic
organization of the gene, -internexin is clearly a member of the
class IV intermediate filament proteins, along with the
neurofilament triplet proteins NF-L, NF-M, and NF-H (Ching and Liem,
1991) and Nestin (Shaw, 1998). In the developing mammalian nervous
system, -internexin mRNA and protein are expressed earlier and more
abundantly than the neurofilament triplet proteins (Kaplan et al.,
1990; Fliegner et al., 1994; Chien et al., 1996, 1998; Giasson and
Mushynski, 1997). Because of its early expression, -internexin may
help stabilize neurons and their processes and provide a scaffolding for the coassembly of the other IF proteins during development. In the
mature nervous system, -internexin protein is found primarily in the
CNS (Chiu et al., 1989), showing a distribution pattern restricted to
neurons, partially overlapping but distinct from that of the
neurofilament triplet proteins. Although many larger neurons express
-internexin along with all three neurofilament triplet proteins, in
some mature neurons, -internexin is the only intermediate filament
protein expressed (Chien et al., 1996).
The -internexin protein has been relatively poorly studied to date,
and little is known about the role of this protein during neuronal
injury and regeneration in the mammalian nervous system. However, clues
regarding its function come from regeneration studies in amphibians and
fish in which xefiltin and gefiltin, respectively, have been found to
be heavily upregulated within developing and regenerating optic nerve
axons (Glasgow et al., 1994; Zhao and Szaro, 1997a,b). One of the
present authors has argued that these two proteins, although somewhat
different in primary amino acid sequence, are in fact lower vertebrate
homologs of mammalian -internexin (Shaw, 1998). If this were the
case, one would expect -internexin to be also upregulated during
neuronal regeneration, as seen for xefiltin and gefiltin. The present
study was therefore conducted to investigate expression patterns of
-internexin in the mammalian CNS using rodent models of peripheral
and central axotomies that have different regenerative outcomes.
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MATERIALS AND METHODS |
Animals and surgery. Male Sprague Dawley rats
(Harlan Sprague Dawley, Indianapolis, IN), 150-175 gm, were
used (n = 4) for each experimental design. Animals were
housed in the McKnight Brain Institute animal facility. For facial
nerve lesions, rats were anesthetized using isoflurane to expose the
right facial nerve near its exit from the stylomastoid foramen. For
crush lesions, the nerve was crushed once with a pair of fine forceps
for 10 sec, ~2 mm from the stylomastoid foramen (~12-14 mm from
the facial nucleus in the brainstem). For transection lesions, the
nerve was isolated from the surrounding tissue and cut at the same
location as the crush. For nerve resection lesions, a 2-3 mm section
of the nerve was removed. After surgery, the wound site was closed using surgical staples, and animals were allowed to recover for 1, 3, 5, 7, 14, or 28 d post-lesion (dpl). Animals were killed by
an injection of sodium pentobarbital (32 mg/kg) via transcardial perfusion with saline, followed by 4% buffered paraformaldehyde. Brains were removed, postfixed in 4% buffered paraformaldehyde for 2 hr, and stored in PBS at 4°C until processing for
immunohistochemistry. Alternatively, brains used for in situ
hybridization were perfused with saline only, rapidly frozen in liquid
nitrogen, and stored at  80°C until use. Contralateral unoperated
facial motor nuclei served as a control.
Axonal transport was blocked using 1.5 mM solution of
colchicine diluted in sterile PBS. Gelfoam was presoaked in either
colchicine or saline before implantation. Under isoflurane anesthesia,
the facial nerve was carefully dissected away from the surrounding connective tissue, and the soaked piece of Gelfoam was placed around
the nerve. The wound was closed, leaving the Gelfoam in place. Animals
were killed at 7 or 14 dpl and processed for immunohistochemistry. Axonal activity was blocked in a similar manner with tetrodotoxin, diluted to 1 µg/ml, and placed around the exposed facial nerve. Animals for these experiments were killed at 3, 5, and 7 dpl and processed for immunohistochemistry.
Rubrospinal tractomies were performed on adult rats deeply anesthetized
with a subcutaneous injection of xylazine (10 mg/kg), followed by
ketamine (100 mg/kg, i.p.). After exposure of the vertebral column at
the C4/C5 level, a dorsolateral funiculotomy was performed on the right
side by inserting a number 11 scalpel blade between the C4 and C5
vertebrae. The wound was closed by suturing the muscle layers and
stapling the skin. Animals were allowed to recover on a heating pad.
Immunohistochemical staining. The -internexin antibodies
were generated in the Shaw laboratory at the University of Florida, and
both are available commercially. They were 2E3 monoclonal and R35
affinity-purified polyclonal antibodies, and their characterization has
been described previously (Evans et al., 2002). Sections through the
facial and red nucleus were cut using a vibratome. Serial sections
50-µm-thick were taken through the entire nucleus and stained for
-internexin. Briefly, sections were rinsed with a 3%
H2O2-PBS solution,
blocked, and incubated with primary antibodies overnight at 4°C
(dilutions were 1:50 for 2E3 monoclonal antibody ascites preparation
and 1:10 for R35 affinity-purified polyclonal antibody at 10 µg/ml).
Appropriate biotinylated secondary antibodies were added (Vector
Laboratories, Burlingame, CA) and linked with avidin-HRP (Vector
Laboratories). Sections were developed using diaminobenzidine,
mounted, dehydrated, and coverslipped.
Retrograde labeling with fluorogold. For retrograde
labeling, 10 µl of a 10% fluorogold solution (fluorogold diluted in
sterile PBS) was injected into two different sites of the whisker pad (5 µl each) 3 d before the termination of each time point.
Sections were processed as above, stained for -internexin, and
visualized using an anti-mouse Texas Red secondary antibody (Molecular
Probes, Eugene, OR).
Stereology. To assess the changes in -internexin
expression, immunopositive cells within the facial nucleus were counted using stereological techniques based on the physical dissector method
described by Coggeshall (1992). Systematic random sections were
selected throughout each nucleus. With the aide of an microcomputer imaging device (MCID) Image Analysis System (Imaging Research, St. Catharine's, Ontario) attached to a light microscope, the entire
facial nucleus was digitized to allow for cell counting and volumetric
analysis. Only neurons whose nuclei were clearly visible were counted.
Significant differences between time points and control were determined
by a one-way ANOVA, followed by a Fisher's PLSD test.
In situ hybridization. Coronal sections (12 µm) through
the facial nucleus were cut for in situ hybridization on a
cryostat and thaw mounted on Fisher Scientific (Houston, TX)
Superfrost/plus slides. All sections were fixed in fresh 4% buffered
paraformaldehyde for 5 min at 4°C, rinsed in PBS, dehydrated through
graded alcohols, and stored in 95% EtOH at 4°C until use.
Oligonucleotide cDNA probes (45-mer) were constructed from the
full-length coding sequence of rat -internexin (GenBank
accession number 019128; residues 1541-1585; CAC CAA CGA GTA CAA GAT
CAT CCG CAC TAA CGA GAA GGA GCA GCT). Control probes consisted of sense
sequences. Probes were 3' end labeled using
[35S]ATP (NEN, Boston, MA) and terminal
transferase. Tissue was hybridized with labeled probe diluted in
hybridization buffer overnight at 42°C in a humidified chamber.
Sections were washed at 60°C in 1× SSC and then 0.5× SSC and
air dried. Sections were then apposed to  -max film (Amersham
Biosciences, Arlington Heights, IL) and stored at room temperature or
dipped in LM-1 emulsion (Amersham Biosciences) and stored at 4°C.
Both were exposed for a maximum of 14 d. Film and emulsion-dipped
slides were developed with Kodak D-19 (Eastman Kodak, Rochester, NY)
and rapid fixer. Films were analyzed using an MCID Image Analysis
System (Imaging Research) attached to a light illuminator. Raw optical
density was determined by outlining the facial nucleus on the control
and lesion side and measuring the light density output. Significant
differences between lesion and unlesioned nuclei were determined by a
one-way ANOVA, followed by a Fisher's PLSD test.
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RESULTS |
Behavioral observations
The motor portion of the facial nerve innervates the whisker pads
of the rat, which are responsible for whisker movement. Lack of whisker
movement therefore reliably indicates that the facial nerve has been
lesioned subsequent to surgery. In all injury paradigms, the whiskers
were immobile after surgery. Recovery of movement after crush and
partial recovery after transection was observed by 28 dpl, indicating
successful reinnervation. In contrast, no movement was observed in the
resection paradigm at 28 dpl. After local application of colchicine to
the facial nerve whisker, movement was lost within 24 hr after surgery.
-Internexin expression after crush, transection, and resection
of the facial nerve
Immunoreactivity for -internexin was completely absent in the
perikarya of uninjured motoneurons, the inner genu, and the peripheral
facial nerve (Fig. 1A).
However many -internexin-positive fibers were seen close to the
facial neurons (Fig. 1), and, within the CNS, numerous
-internexin-immunoreactive fibers and fiber tracts were seen. After
facial nerve transection, strongly -internexin-immunopositive motoneuron perikarya were first seen on the operated side by 3 dpl, and
these continued to increase with time, reaching maximal numbers by 7 dpl (Fig. 1F). This was a dramatic response, and it
was always obvious which side of the section contained the lesioned
nucleus and which was the control. Thereafter, the number of
-internexin-positive cells decreased, and most motoneurons had lost
immunoreactivity by 28 dpl. Axons proximal to the axotomy and at the
axotomy site itself did not show any staining at any time points
examined.
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Figure 1.
-Internexin immunoreactivity after facial nerve
transection. A, Normal unoperated facial nucleus shows
no staining of motoneurons, but immunopositive fiber bundles are
abundant (arrows). B, Seven days after
axotomy, most motoneurons show intense immunoreactivity.
C, D, At 14 and 28 d after injury,
numbers of immunoreactive motoneurons decline. E, High
magnification of -internexin-positive motoneurons at 5 dpl. Note the
staining of the neuropil and cellular cytoskeleton. Some motoneurons
are swollen and show an eccentrically placed nucleus indicative of
chromatolysis. F, Total numbers of
-internexin-positive cells in the facial nucleus after transection
and crush. Maximal numbers are seen at 7 dpl in both lesion paradigms,
with a decline by 28 dpl. Note the fewer -internexin-positive cells
in the less severe (crush) injury paradigm, with a dramatic decline in
numbers that approaches control levels by 28 dpl. Significant
differences between groups is denoted by asterisks.
Significance determined by an ANOVA; p  0.05. Magnification: A-D, 117×; E,
585×.
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After facial nerve crush, staining for -internexin was first noted
in neuronal perikarya by 3 dpl, and maximal numbers were, once
again, observed at 7 dpl (Fig. 1F). Total
immunoreactive cells declined rapidly until 28 dpl, when very few cells
remained labeled. In general, the number of immunopositive cells at
each time point in the crush paradigm was less than seen in
the transection injury paradigm (Fig. 1F). No
staining for -internexin was noted at any time in motoneurons of the
contralateral unoperated facial nuclei or in their axons traveling
within the brainstem in any of the lesion paradigms.
Nerve resection, which created a large gap to prevent regeneration, did
not result in recovery of whisker movement by the end of the
experimental period (28 dpl), showing that motor axons had failed to
reinnervate their targets. Immunoreactivity for -internexin remained
high in these animals through 28 dpl, with maximal numbers of
motoneurons stained (Fig. 2). The
immunocytochemical results obtained with monoclonal antibody 2E3
and affinity-purified polyclonal antibody R35 made against
full-length -internexin were indistinguishable in all of these
experiments. This suggests that the rapid and dramatic increase in
-internexin staining seen in facial neuron perikarya was
attributable to changes in protein expression and not attributable to
alterations in protein processing by post-translational
modification.
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Figure 2.
Comparison of -internexin expression at 28 dpl
in crush (A), transection
(B), and resection (C)
paradigms. No -internexin-positive neurons are seen after crush
injury at 28 d, although a few can still be found after
transection. Nerve resection, which prevents regeneration, results in
sustained upregulation of -internexin. D,
Stereological counting at 28 dpl. Significant differences between
groups is denoted by asterisks. Significance determined
by ANOVA; p 0.05.
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In situ hybridization
To confirm that the upregulation at the protein level was mirrored
at the mRNA level, we examined -internexin mRNA expression by
in situ hybridization at 7 and 14 d after transection.
As expected, -internexin mRNA was expressed strongly in regions rich
in neuronal cell bodies and especially strongly in the cerebellar
granular layer, arguing that the probe used was specific for
-internexin mRNA (data not shown). At 7 d after injury,
-internexin mRNA was clearly upregulated on the lesion side relative
to contralateral control side (Fig. 3).
Surprisingly, we also noted the presence of lesser amounts of mRNA
within motoneurons on the unlesioned side, although these cells showed
a complete lack of immunoreactivity. At 14 d after injury, mRNA
expression was still increased on the lesion side when compared with
control.
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Figure 3.
In situ hybridization for
-internexin in the facial nucleus after nerve transection.
A, C, Contralateral uninjured sides at 7 and 14 dpl, respectively. B, D,
Ipsilateral axotomized sides at 7 and 14 dpl, respectively. Note the
greater grain density over lesioned motoneuron perikarya.
Magnification, 234×. E, Quantitative densitometry of
in situ hybridization signal of facial motoneurons at 7 and 14 d after transection. Note the significantly increased raw
optical density (ROD) measurements of lesioned
motoneurons at both time points (asterisks) when
compared with the contralateral control. Significance determined by
ANOVA; p 0.05.
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Retrograde transport after injury
To establish that target reinnervation of motor axons correlated
with downregulation of -internexin expression, facial nerves were
crushed, and the animals were allowed to recover for 7 or 10 d.
Whisker pads on both sides were injected with fluorogold 3 d
before the animals were killed to determine which facial motoneurons were reconnected with the muscles of the whisker pads by showing retrograde transport of fluorogold. At 7 dpl, no fluorogold was observed in the injured facial nerve nucleus but was present in the
contralateral (control) side. By 10 dpl, fluorogold-positive motoneurons could be seen on the injured side. (Fig.
4). Neurons that were labeled with
fluorogold showed little or no -internexin immunoreactivity,
indicating that these motoneurons had successfully reinnervated to
their target and concomitantly downregulated -internexin protein
production. In contrast, neurons showing strong perikaryal -internexin immunoreactivity showed no fluorogold labeling.
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Figure 4.
Double labeling for retrogradely transported
fluorogold and for -internexin immunoreactivity at 10 d after
crush injury. A, B, Motoneurons that have
not yet reconnected cannot take up fluorogold and remain -internexin
positive. C, D, Fluorogold uptake is
coincident with the disappearance of -internexin immunoreactivity,
showing that target reinnervation results in cessation of
-internexin translation. Magnification, 585×.
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Axonal transport and activity blockage
Colchicine is a plant alkaloid that binds to tubulin, inhibiting
the assembly and promoting the disassembly of microtubules, ultimately
halting axonal transport when applied topically to nerves. To determine
whether retrogradely transported, modulatory signals from the periphery
were responsible for downregulating -internexin protein expression
in uninjured motoneurons, colchicine was applied topically to the
facial nerve. Seven days after application, -internexin
immunoreactivity was increased to maximal levels very similar to what
was seen after physical transection (Fig. 5). In contrast, control animals that
received topical application of saline did not show any upregulation of
-internexin immunoreactivity.
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Figure 5.
-Internexin immunoreactivity in facial nucleus
7 d after application of colchicine or tetrodotoxin to the
peripheral facial nerve. A, Contralateral control
nucleus shows lack of immunoreactivity in motoneurons
(arrows). B, Colchicine-treated
motoneurons (arrows) show a dramatic upregulation of
-internexin staining similar to that seen after nerve transection.
Contralateral control nucleus (C) and
tetrodotoxin-treated facial nucleus (D). Note the
lack of immunostaining on both the control and treated sides
(arrows). Magnification, 234×.
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Because it is possible that a lack of neuronal activity alone could
cause an upregulation of -internexin, we treated the peripheral
facial nerve with tetrodotoxin to block nerve impulses. In these
studies, there was no upregulation of -internexin
immunoreactivity at any time point (Fig. 5); however, retrograde
transport was maintained, as noted by fluorogold-positive neurons in
the facial nucleus of the treated nerve (data not shown).
-Internexin protein and mRNA expression after
rubrospinal tractotomy
To compare the pattern of -internexin expression in a
regenerating system with that in a nonregenerating system, we performed central axotomies involving transection of the rubrospinal tract. Unlike normal motoneurons, normal rubrospinal neurons showed
constitutive, low-intensity immunoreactivity for -internexin (Fig.
6A,C).
After rubrospinal tractotomy, an increase in -internexin
immunoreactivity was observed in axotomized rubrospinal neurons 7 d after injury relative to the contralateral unoperated red nucleus
(Fig. 6B). However, increased immunoreactivity in
axotomized rubrospinal neurons was no longer detectable 14 d after
axotomy, and the intensity of -internexin immunoreactivity was
similar to that seen in contralateral uninjured cells (Fig.
6D). In situ hybridization analysis
revealed a significant upregulation of -internexin mRNA on the
lesioned side when compared with the contralateral control (Fig.
6E). This upregulation persisted at 14 d after
axotomy.
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Figure 6.
-Internexin immunoreactivity and mRNA in
the red nucleus after transection of rubrospinal axons in the cervical
spinal cord. A, C, Ipsilateral control
red nucleus at 7 and 14 dpl, respectively. Note the constitutive
expression of -internexin in unlesioned rubrospinal neurons
(arrows in A and C).
B, D, Contralateral lesioned red nucleus
at 7 and 14 dpl, respectively. -Internexin immunoreactivity is
increased 7 d after tractotomy (arrows in
B) but returns to normal by 14 d after tractotomy
(arrows in D). Magnification, 234×.
E, Quantitative densitometry of in situ
hybridization signal of axotomized rubrospinal neurons at 7 and 14 d after tractotomy. Note the significantly increased raw optical
density (ROD) measurements of lesioned neurons when
compared with the contralateral control at both time points
(asterisks). Significance determined by ANOVA;
p 0.05.
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DISCUSSION |
Using three different facial nerve lesion paradigms, we found that
-internexin protein expression, normally undetectable immunocytochemically in the perikarya of facial neurons, dramatically increases in a transient manner that parallels the rate of muscle reinnervation. This marked change in immunoreactivity was seen with
both monoclonal and polyclonal antibodies, suggesting that it is
attributable to strong induction of expression of -internexin protein and not attributable to, for example, post-translational modification of this protein. Blockage of axonal transport with colchicine also induced perikaryal -internexin expression and suggests that the increase in -internexin protein expression is
regulated by a retrogradely transported signal from the periphery. In
addition, fluorogold injections, which allowed us to examine the
exclusivity of -internexin expression to those neurons that had not
yet reinnervated their targets, further support the idea that an
inhibitory signal from the periphery, specifically from the muscle, may
be responsible for the lack of -internexin expression under normal,
noninjury conditions. It appears that this inhibitory signal works at
the translation level, because mRNA for -internexin is seen in
uninjured facial neurons. This constitutive inhibition by
muscle-derived, retrogradely transported factors, which is absent from
intrinsic CNS neurons, represents a novel mechanism that may explain,
in part, the robust regenerative potential of motoneurons.
In contrast to our findings with -internexin, the neurofilament
triplet proteins NF-L, NF-M, and NF-H are strongly expressed by
uninjured motoneurons but are downregulated after axonal injury (Goldstein et al., 1988; Tetzlaff et al., 1988, 1991; Muma et al.,
1990). The present data therefore suggest that -internexin serves a
function different from the neurofilament triplet proteins. Clues that
-internexin may play a role in mediating neuronal regeneration come
from a number of different observations. Studies in lower vertebrates
with vigorous CNS regenerative capacities, such as amphibians and fish,
have shown that successful regeneration of optic axons is associated
with increased expression of neuronal intermediate filaments proteins
termed xefiltin and gefiltin (Glasgow et al., 1994; Zhao and Szaro,
1997a; Niloff et al., 1998). These proteins are likely the
amphibian and fish homologs of mammalian -internexin (Shaw, 1998).
Although they are somewhat divergent from mammalian
-internexin in primary sequence, comparison of different mammalian
-internexin sequences shows a much greater cross-species sequence
variability than seen with, for example, vimentin. Mammalian and fish
homologs would therefore be expected to be even more divergent in amino
acid sequence, as is the case with xefiltin and gefiltin. In addition,
the distributions of these two proteins parallel the distribution of
-internexin in mammalian embryos (Glasgow et al., 1994; Zhao and
Szaro, 1997b). Furthermore, the draft human genomes reveal no protein
closer to xefiltin or gefiltin in primary amino acid sequence than
-internexin, and no closer homolog of mammalian -internexin has
been observed in these two species. The strong upregulation of an
-internexin homolog therefore appears to be a conserved part of the
regenerative response in vertebrates.
-Internexin is the first neurofilament protein to be expressed
during development, preceding the appearance of the neurofilament triplet proteins (Kaplan et al., 1990), suggesting that it may serve as
a scaffold for assembly of other neurofilament proteins. As development
continues, levels of -internexin mRNA decline, whereas those of NF-L
and the other triplet proteins increase (Fliegner et al., 1990). The
observed upregulation of -internexin we have seen therefore likely
reflects a recapitulation of developmental events and parallels several
other responses seen in injury, including the downregulation of the
neurofilament triplet proteins. It is possible that, to rebuild severed
motor axons, increased amounts of a scaffolding protein are required
before neurofilament triplet protein assembly can occur. Our data in
the rubrospinal system show that -internexin protein expression is
increased only transiently and not as robustly in axotomized central
neurons compared with motoneurons, and it is of course suggestive that
these axons do not normally regenerate.
These results therefore correlate well with those from other
investigators who have found that, although rubrospinal neurons initially upregulate certain "regeneration-associated genes," such
as actin and tubulin, these central neurons abort the regenerative effort and return to preinjury levels, although reconnections have not
been made (Tetzlaff et al., 1994; Fernandes et al., 1999). It appears
that -internexin is regulated in the same manner during regeneration
as actin and tubulin. Other proregenerative molecules, such as
growth-associated protein-43 and type 1 tubulin, remain elevated (Tetzlaff et al., 1991; Linda et al., 1992), despite the fact
that rubrospinal neurons undergo atrophy and show a decline in total
mRNA (Barron et al., 1977, 1989; Tetzlaff et al., 1991, 1994).
In a recent study, Levavasseur et al. (1999) generated
-internexin knock-out mice and showed that these develop normally to
adulthood without any obvious behavioral or anatomical
abnormalities and produced normal offspring. Motor axons from L4
ventral roots were of normal number and size and appeared identical to
those in wild-type animals, and neurofilament triplet protein
containing structures appeared normal at the ultrastructural level.
These studies therefore suggest that -internexin does not have an
essential, nonredundant role in the assembly of the cytoskeleton or for
axonal growth during development. It is however possible that
-internexin has important but subtle and so far overlooked functions
not revealed by assays performed to date or that other molecules can
functionally compensate for lack of this gene product. The specific
role of -internexin upregulation in regeneration has not so far been addressed in these knock-out mice, and it is possible that, in the
absence of this protein, regeneration may be perturbed, an issue that
should be easily addressed experimentally. Interestingly, the level of
-internexin must be tightly regulated because overexpression of
-internexin protein at two to three times the normal level produces
mice with motor coordination deficiencies (Ching et al., 1999). These
behavioral changes correlated with swollen Purkinje cell axons in the
cerebellum and abnormal organelles in large pyramidal neurons of the
neocortex and thalamic neurons. As these animals aged, increased
neurodegeneration was noted similar to that seen in a variety of
neurodegenerative diseases.
The class III neuronal intermediate filament protein peripherin, like
-internexin, is expressed early during development but decreases in
level of expression during the postnatal time period (Escurat et al.,
1988, 1990). Also similar to -internexin, peripherin expression in
affected neurons increases after peripheral nerve injury and decreases
once reinnervation occurs (Oblinger et al., 1989; Terao et al., 2000).
Blocking nerve regeneration or retrograde transport with vincristine
causes peripherin protein and mRNA to remain elevated, supporting the
idea that an inhibitory signal from target tissue may regulate the
expression of peripherin in motoneurons (Terao et al., 2000). Possibly
peripherin and -internexin expression are controlled by the same
peripheral factor or factors. The identity of these putative peripheral
signals and their mode or modes of action remain subjects for
additional study.
It has been theorized that metabolic changes by injured Schwann cells
or a change in Schwann cell-axon connections may provide a signaling
mechanism to the cell body to upregulate and downregulate various
neuronal proteins after injury. A cold block that inhibited fast axonal
transport without inducing an inflammatory response increased the
expression of type 1 tubulin and p75 neurotrophin receptor mRNA in
motoneurons (Wu et al., 1993), as well as increased the major myelin
protein P0, without disrupting the Schwann
cell-axon interaction (Wu et al., 1994). In addition, application of
colchicine or vincristine to uninjured nerve, which halts axonal
transport by disrupting the microtubule complex, can decrease choline
acetyltransferase (ChAT) activity without physical nerve injury
(Bussmann and Sofroniew, 1999; Terao et al., 2000). Other studies using
nerve crush, cut, or ligature also show the same effects on ChAT
production (Rende et al., 1995; Bussmann and Sofroniew, 1999; Terao et
al., 2000). Retrograde signals from the target tissue were interrupted
in all cases, suggesting that ChAT production is also controlled by
axonal transport of an as yet unidentified substance or substances that
do not originate from injured Schwann cells. Based on our results and
the results of Terao et al. (2000), we believe that an inhibitory
signal from the target tissue, in this case, the facial muscles, may be
responsible for suppressing -internexin protein translation in the
adult. After nerve transection or crush, -internexin expression
decreases by 28 dpl, either with or without complete Schwann cell
repair. However, after nerve resection, which prohibits nerve
regeneration, -internexin expression does not decrease by 28 dpl,
suggesting that the modulating signal to upregulate -internexin is
not produced by Schwann cells. Therefore, only after a complete block
of retrograde transport, by either chemical blockage or physical
separation, is the inhibitory signal lost causing -internexin
protein to become expressed. Until axons are reconnected, the
retrograde signal from the periphery cannot reach the motoneuron cell
body and therefore cannot suppress -internexin expression.
Presumably, this tight regulation of -internexin expression in the
normal and regenerating neuron reflects a role for this protein in
nerve regeneration.
| |
FOOTNOTES |
Received Dec. 12, 2001; revised March 22, 2002; accepted March 28, 2002.
This work was supported in part by funds from the State of Florida
Brain and Spinal Cord Injury Rehabilitation Fund and by the American
Heart Association (Florida and Puerto Rico Affiliate). Special thanks
to Lori L. White for her technical assistance.
Correspondence should be addressed to Dr. Wolfgang J. Streit,
Department of Neuroscience, P.O. Box 100244, Building 59, University of
Florida College of Medicine, 100 Newell Drive, Gainesville, FL 32611. E-mail: streit{at}mbi.ufl.edu.
| |
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