The differential expression patterns of messenger RNAs encoding Nogo-A and Nogo-receptor in the rat central nervous system.

Abstract

Nogo-A and Nogo-receptor have been considered to play pivotal roles in controlling axonal regeneration and neuronal plasticity. We investigated the total distribution of Nogo-A and Nogo-receptor mRNAs in the adult rat central nervous system using in situ hybridization histochemistry. Nogo-A is abundantly expressed in both neurons and oligodendrocytes throughout the central nervous system. Interestingly, we could not find any neuron which lacks Nogo-A mRNA expression, indicating that Nogo-A mRNA is universally expressed in all neurons. In contrast, Nogo-R mRNA expression was very restricted. Nogo-R mRNA was expressed in the olfactory bulb, hippocampus, tentia tecta, some amygdala nuclei, cerebral cortex, some thalamic nuclei, medial habenular, whereas we could not detect it in the other regions. Interestingly, we did not detect Nogo-R mRNA in monoaminergic neurons, which are known to have high regenerative capacity, in the substantia nigra, ventral tegmental area, locus caeruleus, and raphe nuclei. In addition, although neurons in the reticular thalamus and cerebellar nuclei are also known to show high capacity for regeneration, Nogo-R mRNA was not detected there. These data indicate that Nogo-A and Nogo-R mRNAs were differentially expressed in the central nervous system, and suggest that the lack of Nogo-R expression in a given neuron might be necessary to keep its high regenerative capacity.

Full text

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THE DIFFERENTIAL EXPRESSION PATTERNS OF MESSENGER
RNAs ENCODING NOGO-A AND NOGO-RECEPTOR IN THE RAT
CENTRAL NERVOUS SYSTEM

Tomohiko Hasegawaa, Koji Ohnob, Michio Sanoa, Takao Omuraa, Kumiko
Omuraa, Akira Naganoa, and Kohji Satob＊

Departments of aOrthopedic Surgery, and bAnatomy and Neuroscience,
Hamamatsu University School of Medicine, Hamamatsu, Japan

The number of text pages of the whole manuscript: 23
The number of figures: 7 (7 pages)
The number of table: 1 (2 pages)





＊Correspondence to: K. Sato
Department of Anatomy & Neuroscience
Hamamatsu University School of Medicine
1-20-1 Handayama, Hamamatsu,
Shizuoka 431-3192, Japan

Tel & Fax: 81(Japan)-53-435-2288
E-mail: Ksato@hama-med.ac.jp

Acknowledgement: Parts of this work was supported by the Ministry of
Education, Science and Culture of Japan and the Ministry of Health, Labor
and Welfare of Japan.

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Abstract

Nogo-A and Nogo-receptor have been considered to play pivotal roles in
controlling axonal regeneration and neuronal plasticity. We investigated the
total distribution of Nogo-A and Nogo-receptor mRNAs in the adult rat
central nervous system using in situ hybridization histochemistry. Nogo-A is
abundantly expressed in both neurons and oligodendrocytes throughout the
central nervous system. Interestingly, we could not find any neurons which
lack Nogo-A mRNA expression, indicating that Nogo-A mRNA is
universally expressed in all neurons. In contrast, Nogo-R mRNA expression
was very restricted. Nogo-R mRNA was expressed in the olfactory bulb,
hippocampus, tentia tecta, some amygdala nuclei, cerebral cortex, some
thalamic nuclei, medial habenular, whereas we could not detect it in the
other regions. Interestingly, we did not detect Nogo-R mRNA in
monoaminergic neurons, which are known to have high regenerative
capacity, in the substantia nigra, ventral tegmental area, locus caeruleus, and
raphe nuclei. In addition, although neurons in the reticular thalamus and
cerebellar nuclei are also known to show high capacity for regeneration,
Nogo-R mRNA was not detected there. These data indicate that Nogo-A and
Nogo-R mRNAs were differentially expressed in the central nervous system,
and suggest that the lack of Nogo-R expression in a given neuron might be
necessary to keep its high regenerative capacity.

Theme: development and regeneration
Topic: regeneration

Key words: in situ hybridization, Nogo-A, Nogo-66 Receptor, CNS myelin
inhibitor, regeneration.

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Introduction

Nogo-A has been discovered as a protein in myelin, which impedes
axonal regeneration [3], [4], [7], [18]. This protein is expressed by central
nervous system (CNS) myelin-forming oligodendrocytes but not by
peripheral Schwann cells [8], [11], [25], and can be observed in
immunoelectron micrographs at the innermost adaxonal and outermost
myelin membranes [8], [25]. This expression pattern is well coincident with
the fact that in the adult CNS axon regeneration is highly restricted [19]. In
addition to its localization in oligodendrocytes, Nogo-A is also expressed in
a range of central and peripheral neurons [8], [11], [25], strongly suggesting
that this protein has additional functions in the CNS. Nogo-A is divided into
three regions separated by two hydrophobic segments. Its large N-terminal
domain (N-nogo) and small C-terminal domain (C-nogo) reside in the
cytoplasmic space, whereas a short stretch of 66 amino acids (Nogo-66)
forms an extracellular loop. Interestingly, both N-nogo and Nogo-66 have
been reported to inhibit axons in vitro independently [4], [5], [6].
Recently, Nogo-66 receptor (Nogo-R) has been identified [4], [7].
Transfection of Nogo-R gene into retinal ganglion cells at a developmental
stage when they otherwise are unresponsive to Nogo-66 promotes growth
cone collapse in response to exogenously applied Nogo-66 [4]. As Nogo-R
is a glycosyl posphatidylinositol (GPI) anchored protein, it needs the
interaction of p75, which is known as the receptor for the neurotrophin
family of nerve growth factor [24]. In addition, Mi et al. have reported that a
new protein LINGO-1 is a component of the Nogo-R/p75 signaling complex,
and plays a role to modulate intracellular Rho activity, which is important in
cytoskeletal regulation [15]. Although Nogo-R is not completely dependent
on Nogo-A, the interaction of both proteins is considered to play pivotal
roles in the inhibition of axon regeneration and other additional phenomena
in the CNS.
The distributions of Nogo-A and Nogo-R have been reported using
immunohistochemisty and in situ hybridization [4], [8], [9], [11], [12], [13],
[21], [25], [26]. Despite the importance of these proteins in the CNS, these
reports mainly deal with their expressions in the restricted areas. In addition,
they are sometimes very contradictory. For example, in the cerebellum,
Fornier et al. and Hunt et al. reported that Nogo-R mRNA was expressed in
Purkinje neurons [4], [9], however Josephson et al. did not detect Nogo-R
mRNA in Purkinje neurons [12]. In addition, Hunt et al. reported that
cerebellar nuclei expressed Nogo-R mRNA [9], whereas Josephson et al. did
not [12]. It is, thus, necessary to perform more wide and detailed
investigations of the expression patterns of Nogo-A and Nogo-R in the adult
rat CNS.

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Materials and Methods

In situ hybridization

Six male Wistar rats (SLC,
approximately 150g were decapitated under diethylether anesthesia. All
experiments conformed to the Guidelines for Animal Experimentation at
Hamamatsu University School of Medicine on the ethical use of animals and
all efforts were made to minimize the number of animals used and their
suffering. The fresh brains and nervous tissues were quickly removed and
Shizuoka,
Japan)
weighing
immediately frozen on powdered dry ice. Serial sections (20μm thick)
were cut on a cryostat, thaw-mounted onto silan-coated slides, and stored at
–80℃. After being warned to room temperature, slide-mounted sections
were fixed in 4% paraformaldehyde in 0.1M phosphatebuffer (pH7.2) for 15
minutes (all steps were performed at room temperature unless otherwise
indicated), rinsed three times (5 minutes each) in 4×SSC (pH7.2) (1×SSC
contains 0.15M sodium chloride and 0.015M sodium citrate), and
dehydrated through a graded ethanol series (70%-100%). The sections were
then defatted with chloroform for 5miniutes, and immersed in 100% ethanol
(twice for 5 minutes each time) before being subjected to hybridization.
Hybridization was performed by incubating the sections with a buffer ［4×
SSC, 50% deionized formamide, 0.12M phosphate buffer (pH7.2),
Denhardt’s solution (Nakarai, Kyoto, Japan), 2.5% tRNA (Roche, Tokyo,
Japan), 10% dextran sulfate (Sigma, Tokyo, Japan)］containing［35S］dATP
(1000-1500 Ci/mmol (37- 555TBq/mmol; New England Nuclear, Boston,
MA, USA))-labeled probes (1-2×d. p. m. /ml, 0.2ml/slide) for 24hour at
41℃. After hybridization, the sections were rinsed in 1×SSC (pH7.2) for
10minutes, followed by rinsing three times in 1×SSC at 55℃ for
20minutes each time. The sections were then dehydrated trough a graded
ethanol series (70-100%). After film exposure for 7 days at room
temperature, the sections were coated with Kodak NBT-2 emulsion (Kodak,
Rochester, NY, USA) diluted 1:1 with water. The sections were then exposed
at 4℃ for 2 weeks in a tightly sealed dark box. After being developed in