Download Regulatory expression of Neurensin-1 in the spinal motor neurons

Neuroscience Letters 421 (2007) 152–157
Regulatory expression of Neurensin-1 in the spinal
motor neurons after mouse sciatic nerve injury
Haruno Suzuki a,1 , Koujiro Tohyama b , Kizashi Nagata a ,
Shigeru Taketani c , Masasuke Araki a,∗
b
a Developmental Neurobiology Laboratory, Department of Biological Sciences, Nara Women’s University, Nara 630-8506, Japan
The Center for Electron Microscopy and Bio-Imaging Research, Department of Neuroanatomy, Iwate Medical University, Morioka, Iwate 020-8505, Japan
c Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan
Received 21 February 2007; received in revised form 29 March 2007; accepted 30 March 2007
Abstract
Axonal regeneration after crush injury of the sciatic nerve has been intensely studied for the elucidation of molecular and cellular mechanisms.
Neurite extension factor1 (Nrsn1) is a unique membranous protein that has a microtubule-binding domain and is specifically expressed in neurons.
Our studies have shown that Nrsn1 is localized particularly in actively extending neurites, thus playing a role in membrane transport to the growing
distal ends of extending neurites. To elucidate the possible role of Nrsn1 during peripheral axonal regeneration, we examined the expression of
Nrsn1 mRNA by in situ hybridization and Nrsn1 localization by immunocytochemistry, using a mouse model. The results revealed that during the
early phase of axonal regeneration of motor nerves, Nrsn1 mRNA is upregulated in the injured motor neuron. Nrsn1 is localized in the cell bodies
of motor neurons and at the growing distal ends of regenerating axons. These results indicate that Nrsn1 plays an active role in axonal regeneration
as well as in embryonic development.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Neurensin; Axonal regeneration; Sciatic nerve; Mouse; Spinal cord
Peripheral axons regenerate after Wallerian degeneration following axonal fiber lesioning. In contrast to the central nervous
system, peripheral nerves have a remarkable capacity to regenerate axonal fibers. Axonal outgrowth after sciatic nerve crush in
rodents has been an excellent experimental model for the study
of axonal regeneration, and numerous neurotrophic factors have
been reported to be involved, including BDNF [25], CNTF [21],
IGF-1 [4] and galectin [5]. In the present study, we utilized this
model in order to elucidate the role of Neurite extension factor1,
Neurensin1 (Nrsn1), during axonal regeneration of peripheral
nerves.
Nrsn1 is a neuron-specific novel protein (previously named
Neuro p-24) that has several membrane domains and one
microtubule-binding domain. This is a unique structure, since
no other membranous protein is known to have a microtubulebinding domain. It is specifically localized in small vesicles of
∗
Corresponding author. Fax: +81 742 20 3411.
E-mail address: [email protected] (M. Araki).
1 Present address: Wakunaga Pharmaceutical Co., Ltd., Research Institute,
Hiroshima, Japan.
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2007.03.077
neurons, and is particularly abundant in neuronal processes [8].
We proposed that Nrsn1 plays certain roles in the transport of
small vesicles to the growing distal end of neurites in association with microtubules, thereby contributing to neurite extension
[1,6]. In cultured neuroblastoma neuro2a cells, the Nrsn1 gene
was upregulated when cells were induced to differentiate into
neurons with retinoic acid, and Nrsn1-immunoreactive vesicles
were concentrated at the growth cones and were often observed
to be fused with the plasma membrane [6]. When COS-7 epithelial cells were transfected with a deletion mutant of Nrsn1
lacking the C-terminal domain, which is believed to contain
an organelle-targeting signal, Nrsn1-immunoreactive vesicles
stayed in the perinuclear region (probably the Golgi region),
indicating that the mutated Nrsn1 protein was not processed
at the Golgi region to exit toward organelles [1]. Furthermore,
in vivo and in vitro pull-down assays confirmed the binding of
Nrsn1 to tubulin [6]. We hypothesize that Nrsn1 has a significant
role in the transfer of membranous vesicles from the trans Golgi
area to microtubule arrays. Thus, Nrsn1 is considered to play
an essential role in neurite extension. Since no other membranous proteins distributing specifically in the nervous tissues has
H. Suzuki et al. / Neuroscience Letters 421 (2007) 152–157
been reported to have a functional microtubule-binding domain,
this unique protein Nrsn1 may possibly have a novel function in
neuronal cellular physiology.
We have partially described Nrsn1 localizations in the peripheral nerves: intense Nrsn1 immunoreactivity was found to be
localized in motor nerve axons of the developing mouse during
postnatal development but to decrease gradually after postnatal
day 15, when the axonal innervation of muscle is completed [1].
Dorsal root ganglion cells robustly extend long neurites in vitro
and numerous Nrsn1-immunoreacitve vesicles were observed
in the growing processes [6]. In the present study, to clarify the
role of Nrsn1 in neurite extension of peripheral nerves, we have
extended our study to Nrsn1 expression in regenerating peripheral nerves by paying particular attention to the cell bodies of
the spinal motor neurons. We demonstrated that the Nrsn1 gene
is actually upregulated soon after axonal lesioning and during
axonal regeneration in the axotomized motor neurons.
Sixty-four male mice (ICR) between 60 and 70 days after
birth were used. Mice were anesthetized with an intraperitoneal
injection of pentobarbital and the right sciatic nerve was exposed
at the midgluteal region. The sciatic nerve was crushed three
times for 10 s with fine-tipped forceps that had been chilled by
liquid nitrogen. After various time periods, mice were deeply
anesthetized again with pentobarbital and perfused with fixative.
All animal procedures employed in the study were consistent
with the AAALAC guidelines.
For immunocytochemical staining, animals were sacrificed
by an intraperitoneal injection of excess pentobarbital. They
were perfused transcardially with ice-chilled fixative consisting of 4% paraformaldehyde and 0.3% glutaraldehyde in 0.1 M
potassium phosphate buffer, pH 7.4 (PB), for 5 min. Perfused
spinal cords were immersed in the same fixative without glutaraldehyde for 5–6 h at 4 ◦ C. After thorough washing with PB
containing 20% sucrose, 30 ␮m sections were cut on a cryostat and stored in PBS containing 0.2% Triton X-100 (PBSX).
Immunostaining was carried out under free-floating conditions
using the avidin–biotin–peroxidase complex system (Vectastain
ABC kit: Vector, USA) as described previously [1,8]. Sections were incubated with 2% fetal bovine serum (FBS) in
PBS, followed by an incubation with a primary antibody at a
desired concentration. They were then incubated in turn with
biotin-conjugated anti-rabbit IgG diluted 1:400 and with the
avidin–biotin–complex diluted 1:800. In some cases, sections
of 10 ␮m thickness were placed on gelatin-coated glass slides
and subsequently processed for immunostaining. The primary
antibody was diluted with PBS-Tx containing 2% FBS. The primary antibodies used in the present study were rabbit polyclonal
anti-Nrsn1 [8], mouse monoclonal anti-Nrsn1 antibody (Antip24; BD Biosciences, San Jose, CA), mouse anti-neurofilament
200 (Sigma) and mouse anti-acetylated tubulin (Sigma). In order
to identify microglial cells that gather around injured nerve
cells, rabbit polyclonal anti-TB4 (thymosin ␤-4) [20] was also
used. As controls, the primary antibody was either replaced by
a normal rabbit IgG fraction (Vector, USA), or eliminated.
In situ hybridization was performed as follows: mice were
sacrificed as described above, and perfused spinal cords were
isolated and immersed in the same fixative without glutaralde-
153
hyde overnight at 4 ◦ C. Frozen sections of 30 ␮m thickness
were cut, and processed for in situ hybridization by a freefloating method in a 1.5 ml centrifuge tube. The sections
were washed by PBS containing 0.1% Tween-20 (PBST),
and then treated with 1 ␮g/ml Proteinase K for 10 min and
refixed with 4% PFA containing 0.1% glutaraldehyde for 20 min
at room temperature. Following several washes, the sections
were prehybridized with hybridization solution for 60 min at
68 ◦ C. The hybridization solution contained 50% formamide,
5 × SSC (0.75 M NaCl and 0.075 M sodium citrate), 5 mM
EDTA, 50 mg/ml yeast tRNA, 50 mg/ml heparin, 0.2% Tween20 and 0.5% CHAPS. Digoxigenin-UTP-labeled Nrsn1-specific
probes diluted with hybridization buffer were hybridized to
the sections overnight at 68 ◦ C. Following hybridization, the
sections were washed several times and sequentially treated
with blocking solution 1 (containing 2% Roche Blocking
Reagent) and blocking solution 2 (containing 2% Roche
Blocking Reagent and 20% lamb serum). After blocking, the
sections were treated with alkaline phosphatase-conjugated antidigoxigenin fragments diluted in blocking solution 2 overnight
at 4 ◦ C. After washing, specific hybridization was detected with,
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
substrates.
Nrsn1 localization was examined immunocytochemically in
regenerating sciatic nerves and in cell bodies of ventral horn
motor neurons (Fig. 1). Mice were sacrificed 5, 10, 15, 20 and
30 days after operation. The spinal cords were removed and the
L2 to L5 regions were isolated. Sciatic nerves at the crush site
were also isolated and examined similarly.
The crush site was easily accessed by the presence of surgical
strings that were inserted during the crush operation. Acetylated
tubulin and Nrsn1 were detected at the distal end of regenerating
neurites 10 days after the crush operation (day 10), indicating
that axons were now actively regenerating (Fig. 1A and B). The
distribution of the two molecules did not necessarily coincide
and some neurites contained a high level of acetylated tubulin but only a small number of Nrsn1-positive granules. Nrsn1
localization was less intensely observed at the more proximal
regions of the neurites (data not shown).
When the spinal cord was stained for Nrsn1, some of the
cell bodies in the ventral horn became positively stained for
Nrsn1 on day 5, and became more intensely stained afterwards
(Fig. 1C and F). It appears that most intense immunostaining
was observed in the cell bodies on day 10, and thereafter they
gradually became less stained. Nrsn1 positive cell bodies in the
ventral horn were counted under a microscope, and positive cell
number increased by day 5 and decreased gradually by day 20
(Fig. 1G).
To confirm whether neurons whose cell bodies were intensely
stained for Nrsn1 had actually been lesioned, double immunostaining for both Nrsn1 and thymosin was performed (Fig. 2).
Thymosin is a marker for microglial cells [20]. Numerous
thymosin-positive microglial cells were found in the ventral
horn area, where these cells were observed to surround Nrsn1immunoreactive cell bodies, suggesting that in those injured
cells, presumably motor neurons, the Nrsn1 gene was activated
(Fig. 2C).
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H. Suzuki et al. / Neuroscience Letters 421 (2007) 152–157
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Fig. 2. Double immunostaining of Nrsn1 (red) and thymosin (green) in the spinal cord on day 5 (A and B) and day 15 post-lesioning (C). The area indicated by a
box in A is shown at higher magnification in B. (B) A few Nrsn1-positive dot-like structures are observed in the cell bodies. On day 15, numerous cell bodies contain
Nrsn1-positive structures, some of which are surrounded by thymosin-immunoreactive (green) microglial cells (arrowheads). Bar in B is 10 ␮m, and is applied to C
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
Fig. 3. In situ hybridization for Nrsn1 mRNA expression in the spinal cord. (A) Unoperated mouse, (B) day 5, (C) day 10 post-lesioning and (D–F) show Nrsn1
positive cell bodies in the spinal cord on day 5 at higher magnification. D (lesioned side) and E (unlesioned side) correspond to the areas shown by arrows in B. F
shows positive cells in D at higher magnification. Bar in A is 50 ␮m, and is applied to B and C. Bar in D is 100 ␮m, and is applied to E. Bar in F is 10 ␮m.
Nrsn1 mRNA localization in the spinal cord was then detected
by in situ hybridization, and cells, particularly in the ventral
horn, were positively stained in the spinal cord of lesioned mice
(Fig. 3). There were only a few positively stained cells in spinal
cords of normal mice. In spinal cords of lesioned mice on day
5, numerous cells were found to be strongly positive for Nrsn1
mRNA (Fig. 3D). Mice on day 10 showed fewer cells than had
been seen on day 5, although there were still many cells moderately stained for Nrsn1 mRNA. These observations agreed with
those obtained by immunocytochemistry for Nrsn1.
To see whether other neuron-specific substances were also
upregulated, spinal cords on day 5, 10 and 15 were stained
immunocytochemically for neurofilament 200 and synaptotagmin (Fig. 4). We chose these two proteins because their functions
in axonal regeneration differ from that of Nrsn1: neurofilament
200 is a cytoskeleton protein, while synaptotagmin is a synaptic vesicle associated protein. On day 5 some cells were found
to be moderately stained for neurofilament 200 and on day 10
many cell bodies at the ventral horn were positively stained
both for Nrsn1 and neurofilament 200. On day 15, the cell bodies became intensely stained for synaptotagmin, suggesting that
these proteins are also needed for axonal regeneration.
The present study demonstrated that the Nrsn1 gene is upregulated in motor neurons during peripheral axonal regeneration
Fig. 1. Distribution of Nrsn1 in the regenerating axons of sciatic nerves and the motor nerve cell bodies after sciatic nerve lesioning. (A and B) Growing processes at
the distal end are doubly stained for (A) acetylated tubulin and (B) Nrsn1 on day 10 post-lesioning. Note that the Nrsn1 localization does not necessarily correspond
to that of acetylated tubulin. Processes indicated by arrows are intensely stained for acetylated tubulin but only faintly for Nrsn1. (C) Ventral horn of the spinal cord
at Lumbar (L) 2–3 regions of an unoperated mouse. The same areas of day 5, 10 and 15 post-lesioning are shown in (D–F), respectively. In normal mice, cell bodies
are not stained (arrows in C) with occasional Nrsn1-positive granules (arrowheads) in processes. On day 10, immunostaining of Nrsn1 in the cell bodies becomes
very intense (arrows in E) and persists up to day 15 (F). Bar in A is 10 ␮m and is applied to B. Bar in E is 10 ␮m and is applied to C, D and F. (G) Quantitative
measurement of Nrsn1 positive cell number. Nrsn1 positive cell bodies in the ventral horn at L2 and L3 were counted under a microscope and are shown as positive
cell number on one section. Nrsn1 expression level of the cell bodies was not taken into account.
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H. Suzuki et al. / Neuroscience Letters 421 (2007) 152–157
Fig. 4. Localization of neurofilament 200 (A and B) and synaptotagmin (B and C) in the ventral horn of the spinal cord after lesioning. In the unoperated mouse,
the cell bodies are not stained for neurofilament 200 (A), while on day 10 post-lesioning, cell bodies are intensely stained (arrows in B). In the unoperated mouse,
immunostaining for synaptotagmin is only occasionally seen (C), while cell bodies are more intensely stained on day 15 post-lesioning (arrows in D). Bar in C is
10 ␮m, and is applied to A, B and D.
and that Nrsn1 is actually accumulated in the cell bodies of
lesioned motor neurons and the distal ends of regenerating
axons.
Nrsn1 upregulation in the lesioned cell bodies was confirmed
by both protein and mRNA localization after axonal injury. In the
spinal cord of normal mice, Nrsn1 immunoreactive materials are
found occasionally in neuritic fibers and show a small dot-like
feature. This is a common feature of Nrsn1 immunocytochemical localizations in nervous tissues, such as the cerebral cortex,
hippocampus, and most other areas [1,8,15], since Nrsn1 is normally localized on the small membranous vesicles distributed
mainly in neurites. On day 5 after crush injury, immunoreactive
materials were found in the cell bodies of motor neurons. Intense
staining was then found within the cell bodies on day 10, and still
remained on day 15. Whether the Nrsn1-positive cell bodies are
actually those of the injured motor neurons or not was examined by double staining for Nrsn1 and thymosin, a microglial
marker [20]. Most Nrsn1-positivie cell bodies were surrounded
by thymosin ␤-4 immunoreactive cells and/or processes, and
those less intensely immunoreactive were seldom accompanied
by thymosin-positive cellular components. These observations
suggest that Nrsn1 is upregulated in regenerating motor neurons.
Neurodegenerative disorders are associated with the activation of microglia [12,19] and some studies have also implicated
microglia as potential mediators in neurodegenerative processes
[10,18,24]. The projection of the lumbosacral motor neuron
column at the mid-thigh level of the sciatic nerve is topographic
and microglial cells showed fast activation within the injured
topographic area [9]. Since thymosin ␤-4 is markedly elevated
in the cell bodies and processes of activated microglia [20],
the present observations indicate that those Nrsn1-postivie
cells are actually damaged by axonal lesioning and that Nrsn1
is synthesized in those damaged motor neurons, much more
intensely than in undamaged cells. A peripheral nerve lesion
induces a loss of synapses from the surface of motor neurons in
the spinal cord [2,11], and, after axotomy, activated astrocytes
extend processes between the motor neuron surface and the lost
synapses [3]. Several cell adhesion molecules are considered
to be involved in these events, including nectin, N-cadherin
and NCAM [13,14,23]. Changes in the expression patterns
of nectin, N-cadherin and NCAM have been surveyed in
spinal cord motoneurons after sciatic nerve transection, and
complex changes in the spatiotemporal expression patterns
were reported [26]. Only a few studies have so far been done on
such expression patterns of molecules in motor neurons after
sciatic nerve transection.
In situ hybridization for Nrsn1 mRNA revealed that Nrsn1
production remains at a low level in motor neuron of normal
mice. The message increases markedly soon after axonal crush,
and an intense signal for Nrsn1 mRNA was detected in the cell
bodies of motor neurons on day 5. This mRNA level persisted
for a while and then decreased by day 15 (data not shown).
Nrsn1 protein appeared to be actively synthesized only in the
initial phase. This indicates that membranous vesicles necessary
for axonal regeneration are produced mostly in the early phase
of regeneration and stored in the cell bodies. Nrsn2, a homolog
of Nrsn1, is also localized in axons of the sciatic nerve (unpublished data) and regenerating nerves of dorsal root ganglion
(DRG) cells [16]. It is possible that Nrsn1 and Nrsn2 have different functions in axonal regeneration and that their expression is
temporally regulated during axonal regeneration. Previous studies indicated that myelin sheaths exhibit rapid degeneration after
H. Suzuki et al. / Neuroscience Letters 421 (2007) 152–157
crush lesioning, followed by the initiation of axonal regeneration by 5–10 days [7,17,22]. The distal end of the sciatic nerves
was positively stained for Nrsn1 by day 10 after axonal injury.
This suggests that Nrsn1-positive structures (small membrane
vesicles) are accumulated at the growing distal end of axons by
day 10. In accordance, Nrsn1 mRNA is detected on day 5 and
Nrsn1 immunoreactivity is already present in the cell bodies.
In our previous study, we showed that cultured DRG neurons
extended long branching neurites in which numerous Nrsn1positive vesicles were localized [6]. Antisense oligonucleotide
inhibition of Nrsn1 synthesis resulted in the transient retraction
of neurites in cultured DRG cells and neuro2a cells. The present
study suggests Nrsn1 has some other physiological function in
axonal regeneration. It is completely unknown but important to
understand by what mechanism the Nrsn1 gene is upregulated
in DRG cells and motor neurons when they are injured. During
postnatal development of the mouse, motor nerve axons at the
musculature are initially intensely stained for Nrsn1 and then
gradually become less stained during subsequent development
[1]. In the mature mouse, the motor nerve axons are no longer
stained for Nrsn1, suggesting that the Nrsn1 gene is downregulated once the fiber connection is properly completed. Although
the molecular mechanism of Nrsn1 in neurite extension remains
unknown, the present observations indicate that Nrsn1 synthesis at the initial phase is an important prerequisite for neurite
regeneration. In our future study, it will be important to describe
Nrsn1’s localization in the regenerating motor nerve axons more
precisely and to clarify Nrsn1’s function in the formation of
proper fiber connections.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgments
[18]
We are grateful to Dr. Edith McGeer for her critical comments on the manuscript. A part of this work was supported
by a research grant for frontier science from Nara Women’s
University.
[19]
[20]
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