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Comparison of gene expression of SCG10 and Stathmin/p19 in aging rat brain: an in situ hybridization study

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Comparison of gene expression of SCG10 and Stathmin/p19 in aging rat brain: an in situ hybridization study

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Content Comparison of Gene Expression
of SCG10 and Stathmin/p19 in Aging Rat Brain:
an In Situ Hybridization Study
by
Minghua Cao
A Thesis Presented to the
FACULTY OF THE LEONARD DAVID SCHOOL OF GERONTOLOGY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE IN GERONTOLOGY
May 1995
Copyright 1995 Minghua Cao
UNIVERSITY OF SOUTHERN CALIFORNIA
LEONARD DAVIS SCHOOL OF GERONTOLOGY
University Park
Los Angeles, CA 90089
This thesis, written by
____________M ihG cHULA C/10___________
under the director of h e.Y' Thesis Committee, and approved by all its
members, has been presented to and accepted by the Dean of the Leonard
Davis School of Gerontology, in partial fulfillment of the requirements for
the degree of _ m / K t e r of s q b n c b i n g b r o n t o i o ^
Dean
Date _ r f a n . ( L / f ? S
THESIS CO]
Chairman
Acknowledgments
I have been very fortunate in having had the opportunity to
study in Leonard Davis School of Gerontology, University of
Southern California for the past three years as an international
studen t.
The school director, Dr. David Peterson and associate
director of student affairs, Pauline Abbott have given me many
helps and generous support in both my academ ic life and in
overcoming cultural barrier. I am grateful to them.
This thesis would not have been possible without having
been as a Research Assistant in Dr. Nozomu Mori's lab during my
graduate study in the Leonard School of Gerontology. I am
indebted to Dr. Mori for his valuable instruction and great
encouragem ent while working in his lab.
The special gratitude should be conveyed to Dr. Thomas H.
McNeill, my thesis advisor. His very intelligent guidance and
criticism greatly stimulated my independent thinking.
I would also like to thank Dr. Toshiyuki Himi, my fellow
worker for his valuable technical expertise and helpful
discussion.
And last, but not least thanks to my sister Mingyi Cao for
her unconditional love and support. Her love and support grant me
a ground on which I am able to dream more...
iii
Table of Contents
I. Introduction . . . . . . . 1
A. Objective of the Study . . . . . 1
B. Significance . . . . . . . 2
Role of Neuroplasticity in Aging . . . 2
Role of nGAPs in Neuroplasticity . . . 3
SCG10 and Stathmin/P19 in nGAPs . 5
C. Background Information . . . . . 6
SCG10 Background Information . . . 6
Stathm in/p19 Background Information . 9
Similarities betw een SCG10 and Stathmin/p19 13
Distinctions betw een SCG10 and Stathmin/p19 17
Sum m ary . . . . . . . 21
II. Materials and Methods . . . . . 2 3
Animals and Tissue Sections . . . . 2 3
In situ Hybridization . . . . . . 2 3
Quantitative Analysis of Film Autoradiography 2 4
III. R esults . . . . . . . . 2 6
Significant D ecreases of Stathmin/p19 mRNA
Expression in Hippocampus During Aging 2 6
D ecrease Trends of Stathmin/19 mRNA
Expression During Aging in Several Other
Regions Examined . . . 2 7
Unchanged Stathmin/p19 mRNA Expression
Levels During Aging in Olfactory Bulb
and other brain regions . . . . 2 7
SCG10 mRNA Expression Levels are Primarily
unaffected During Aging in Examined
Brain Areas . . . . . 2 8
IV. D iscussion . . . . . . . 3 6
V. R eferences . . . . . . . 44
VI. A bbreviations 5 4
List of Tables and Figures
Table 1
Table 2
Fig.1
Fig.2
Fig.3
Fig.4
Fig.5
Quantitative Analysis of the Stathmin/p19
mRNA Expression Levels in Various Brain Regions
of Male Fischer 344 Rats a s Determined using
In Situ Hybridization H istochem istry .
Quantitative Analysis of the SCG10 mRNA
Expression Levels in Various Brain Regions
of Male Fischer 344 R ats a s Determined Using
in Situ Hybridization H istochem istry .
In Situ Hybridization of Stathmin/p19 mRNA in
Coronal Sections of Hippocampus of Young and
Old Rat Brains. Computer Processed Film Images
of the Sections Hybridized with 35S -la b e le d
Antisense Stathmin/p19 Riboprobe are Shown
In Situ Hybridization of SCG10 mRNA in Coronal
Sections of hippocampus of Young and Old Rat
Brains. Computer P rocessed Film Images of the
Sections Hybridized with 35S-labeled A ntisense
SCG10 Riboprobe are Shown. The SCG10 Panel
Represents Hybridization Im ages of the Adjacent
Sections of Stathm in/p19 Panel in Fig.1
The Effects of Aging on Stathmin/p19 mRNA
Expression Levels in Hippocampus Section of
Rat Brain . . . . . . .
The Effects of Aging on Stathmin/p19 mRNA
Expression Levels in Olfactory, Striatum,
Mid Brain and Cerebellum Sections of
Rat Brain . . . . . . .
The Effects of Aging on SCG10 mRNA
Expression Levels in Different Regions of
Rat Brain . . . . . . .
V
Abstract
SCG10 and stathmin/p19 are two evolutionary related
neuronal growth-associated-proteins (nGAPs) which are proposed
to play important roles in brain. The expression levels of these
genes were examined in various brain regions of young, middle
aged and old rats by in situ hybridization to evaluate age-related
changes in normal aging. Significant decreases of stathmin/p19
mRNA expression were observed in hippocampus of aged rats,
while the decrease trends were noted in several other regions as
well. In contrast to the selective reduction of stathmin/p19 gene
expression in aging rat brain, the mRNA levels of its neuronal-
specific isoform, SCG10, were unaffected during aging. By
comparing the expression profiles of these two genes with other
nGAPs, the possible dynamics on evolution and aging in nervous
system is explored.
I. Introduction
A. Objective of the Study
SCG10 and stathm in/pi 9 are two evolutionarily related
neuronal growth-associated-proteins which are proposed to play
important roles in adult brain (Okazaki e t al., 1993; Himi e t al.,
1994; McNeill e t al., 1992). As previously observed, though
sharing high identity in their sequences, the gene expression
patterns of these two proteins in adult rat brain are distinct
(Himi e t al., 1 994).
To explore age-related changes th at occur in the brain on
molecular level, and evaluate to which degree the changes
correlate with these two intriguing molecules, we examined the
expression of SCG10 and stath m in/19 mRNA in various brain
regions of young and aged rats by in situ hybridization assays.
B. Significance
Role of Neuroplasticity in Aging
The nervous system m ust adapt to its environment during
the lifetime of the animal. The ability of neurons to adapt or
modify their structure in response to various intrinsic and
extrinsic factors is referred to as "neuroplasticity" (Fishman,
1989; Cotman and Nieto-Sampedro, 1984). Neurons exhibit a
remarkable plasticity of form, both during neural development
and during the subsequent remodelling of synaptic connectivity
(Strittm atter, 1992).
It has been proposed th a t the deterioration in brain
function th at occurs during ageing may be due to a loss of
neuronal plasticity (Cotman and Holets, 1985; Cotman e t al.,
1993; Fishman, 1989; Mori, 1993). There is evidence th at
neuronal plasticity does decline with advancing age (Bartus e t
al., 1982; Hornberger e t al., 1985; Fischer e t al., 1987). Brain
plasticity can be teste d in experimental paradigms of lesion-
induced synaptic remodeling (Cotman and Nieto-Sampedro,
1984). Removal of synaptic connections following a
deafferentation lesion results in a sprouting of remaining
afferents that term inate near th e denervated area. While the
ability to form new synapses in response to injury occurs in
both the young and aged animals, previous studies have
suggested that the injury-induced response in aged animals was
delayed and/or not as extensive as compared to young animals
(Scheff e t al., 1980; Cotman and Holets, 1985).
This raises the questions of how much of the plasticity
remains in later stages of life, and what mechanisms are
involved in this loss of cell plasticity. Answers to this will
lead to an effective search for therapy of memory loss and
delayed recovery from certain neuronal dam age in the aged and
age-related diseased brains.
Role of nG A Ps in Neuroplasticity
To explore the mechanisms of this plasticity on molecular
level, it seem s not inconceivable th a t there is a se t of genes
th a t is especially important to confer the neuronal phenotype,
including structural remodeling (Fishman, 1989) which
continues throughout life, and the nerve cells appear to have
intrinsically programmed growth potentials (Banker and Cowan,
1979).
The current study is based on the hypothesis th a t key
molecules for understanding the mechanisms of loss of neuronal
plasticity would be the so-called neuronal growth-associated
proteins (nGAPs). nGAPs are a group of molecules whose
expression is tightly correlated with neurite outgrowth, which
include but not limited to neurofilaments, peripherin, MAP-2,
tau, GAP-43 (Basi e t al., 1987; Cilmer e t al., 1987) and SCG10
(Mori, 1993; Stein e t al., 1988). The overall expression profiles
of th ese nGAPs are in some ways similar in th a t they are
expressed a t high levels during neurogenesis in embryonic and
postnatal periods, low but significant levels of expression still
persist into adulthood. Thus, the expression of nGAPs is closely
correlated with neurite elongation during neurogenesis, and it
also seem s to play important roles during dendritic
remodelling, which is a structural basis for synaptic plasticity
(Benowitz and Routtenberg, 1987; Benowitz e t al., 1990;
Fishman, 1989; Gispen e t al., 1992; Johnson e t al., 1992; McNeill
e t al., 1992; Nunez e t al., 1988; Pfenninger e t al., 1992;
Strittm atter e t al., 1992; Wong e t al., 1990).
Many of these nGAPs are induced in PC12 cells during
neurite elongation such as seen following nerve growth factor
(NGF) treatm ent (Drubin e t al., 1985; Federoff e t al., 1988;
Lindenbaum e t al., 1988), which su g g ests th a t nGAP expression
may be regulated by extracellular factors including
neurotrophins in the nervous system . If abnormal expression of
certain nGAPs, e.g. neurofilament subunits (NF-L and NF-H), is
introduced in transgenic mice, the mice develop progressive
neurological defects th at resemble the pathology of m otor
neuron disease and amyotrophic lateral sclerosis (Cote e t al.,
1993; Xu e t al., 1993). Thus, nGAPs may play an essential role
in neuronal development and the establishm ent of the nervous
system .
Most nGAPs are phosphoproteins, whose levels of
phosphorylation change in response to extracellular signals such
as NGF or in aging-related neurodegenerative conditions such as
in Alzheimer's disease. Phosphorylation is known to be involved
in synaptogenesis and remodelling in the hippocampus, the
structure which seem s involved in memory function
(Routtenberg, 1984), and memory loss is a frequently occurring
functional deficit of the ageing human brain. On the other hand,
nGAPs are also known to be involved in pathogenesis and/or
reactive sprouting responses in Alzheimer’s disease (AD) and
Parkinson’s disease (PD) (Masliah e t al., 1991a; 1992a; 1992b;
McNeill, 1992).
Thus both the examination of the expression profiles of
nGAPs during ageing and the exploration of regulatory
mechanisms of those genes will contribute to understanding of
molecular mechanisms of loss of neuronal plasticity in the
ageing brain.
SCG10 and Stathmin/p19 in nG APs
Given th at nGAPs are central in understanding the
mechanisms of loss of neuronal plasticity in aging, we aim to
characterize changes in the time course of nGAP gene
expression in order to evaluate age-related changes th a t occur
in the brain in normal conditions. .
In this study we focus on the examination of SCG10 and
stathm in/p19.
As one of the nGAPs, SCG10 seem s to play a role in
neuronal differentiation and structural plasticity (McNeill e t al.,
1992; Mori e t al., 1990; Mori, 1993). It shares an amino acid
sequence similarity with an 18- to 19-kDa phosphoprotein
named stathmin / p i 9, which is more broadly expressed in a
variety of cell types of the neural, immune, and reproductive
system s. The sequence similarity has suggested th at SCG10 and
stathm in/pl 9 have been derived from structurally and
evolutionarily related genes [Okazaki, 1993].
Is the expression of SCG10 and /or stathm in/p19
maintained in the later sta g es of life? By studying these two
homologous genes to g eth er and comparing their expression
profiles with each other we may not only attem p t to understand
the role of a certain nGAP alone, but also the correlations among
these molecules and further, it may lead a way to explore
possible evolutionary dynamics of aging process.
C. Background information
SCG10 Background Information
SCG10 is a neural-specific protein consisting of 179
amino acid residues (Stein e t al., 1988), which was originally
isolated from the rat as a marker of neuronal cells of the
developing neural crest (Anderson and Axel, 1985; Mori e t al.,
1990). It is expressed in various neuronal subtypes in both
central and peripheral nervous system s (Stein e t al., 1988; Mori,
1993; Himi et al., 1993). The m ouse SCG10 protein sequence
differs from the rat SCG10 sequence only by one amino acid
residue (Okazaki e t al., 1993). This strong amino acid sequence
conservation seem s to su g g est th a t SCG10 exerts an important
function in neurons.
The neural-specific expression of the SCG10 gene is
transcriptionally regulated and primarily determined by the
presence of a neural-restrictive silencer in the distal region of
the SCG10 prom oter (Mori e t al., 1990; 1992).
Expression of SCG10 mRNA is observed at high-levels in
developing neurons (Stein e t al., 1988), and persists into
adulthood at low but significant levels (Himi e t al., 1994). It is
induced in PCI 2 cells and adrenal medullary chromaffin cells by
nerve growth factor (NGF) (Anderson and Axel, 1985; Stein e t
al., 1988), and its induction is partially inhibited in the
presence of glucocorticoids (Anderson e t al, 1989a; Stein e t al.,
1988).
SCG10 is also regarded as a marker of neuronal plasticity
and characterized as a growth-associated-protein (GAP) since it
is upregulated during regeneration of injured postganglionic
sym pathetic nerve (Anderson, 1989a; Anderson, 1989b; Mori,
1993). SCG10 is associated with cellular membranes,
transported through axons, and accumulated in the growth cones
of growing neurons in culture (Stein e t al., 1988).
In situ hybridization studies of SCG10 mRNA in the adult
rat brain revealed th a t it is expressed in certain subsets of
neurons, preferentially, it is expressed in neurons with long-
projecting axons and/or extensive dendrites. Positive areas
with the highest level of its expression in the adult brain
include: mitral cells of the olfactory bulb, neurons in the
piriform cortex, pyramidal and granule cells in the hippocampus,
Purkinje and granule cells in the cerebellum, as well as several
nuclei in the brain stem (Himi e t al., 1994). Moreover, SCG10
mRNA expression is not restricted to a single functional
phenotype - It could be expressed in both inhibitory and
excitatory neurons, and so forth. Also, th e expression does not
correlate to specific neurotransm itter phenotypes - it was
expressed a t high levels in noradrenergic, dopaminergic, and
serotonergic neurons. Large neurons tend to express SCG10 at
higher levels than small neurons (Himi e t al., 1994).
The distribution of SCG10 proteins was examined by
immunohistochemistry using antibodies against SCG10. The
results were similar to th at of the in situ hybridization
experiments, although the SCG10 protein was distributed in
wider areas, probably owing to transportation along dendrites
and axons (Sugiura e t al., unpublished).
The distribution pattern of SCG10 indicates th a t the brain
areas where the higher expression was observed in the adult are
known to maintain neuronal plasticity in adults (Mori, 1993),
suggesting SCG10 may be involved in th e remodelling of
synaptic connections in response to various intrinsic and
extrinsic signals in adult brain. In deed SCG10 was shown to be
induced in the contralateral cortex following unilateral striatal
deafferentiation (Cheng e t al., 1991).
The deduced amino acid sequence of SCG10 predicts a
polypeptide of 21 kDa (Stein e t al., 1988) th a t shares a high
degree of amino acid sequence homology (i.e. 74% identity) to a
18- to 19-kDa neural- enriched phosphoprotein, stathm in (Sobel,
1991), suggesting th a t SCG10 is the neural-specific isoform of
this recently identified gene family (Okazaki e t al., 1993),
Furthermore, the variously regulated expression profiles
of SCG10, e.g. developmental regulation (Anderson and Axel,
1985), NGF-inducibility (Anderson and Axel, 1985, Stein e t al.,
1988), glucocorticoid-suppressibility (Stein e t al., 1988),
upregulation during axonal regeneration (Anderson and Axel,
1985) and significant expression in subset neurons of the adult
brain (Himi e t al., 1994), are all reminescent of GAP-43
(Benowitz e t al., 1990; Fishman, 1989; Gispen e t al., 1992;
Pfenninger e t al., 1992; Skene, 1984; Skene, 1989; Strittm atter
e t al., 1992), the well studied neuronal growth-associated
protein (Benowitz and Routtenberg, 1987).
Stathmin/PI 9 Background Information
Stathmin was identified as a ubiquitous, neuron-enriched
phosphoprotein. It is a small (19 kDa), soluble, heat-stable
protein, present in the cytoplasm of cells as several isoforms
(Sobel, 1991).
Stathmin is also designated as pi 9 (Pasmantier e t al.,
1986), Opl 8 (Hailat e t al., 1990), etc. It is widely expressed in
both neuronal and non-neuronal tissues (Amat e t al., 1990; Amat
e t al., 1991; Schubart, 1988; Sobel, 1991; Sobel e t al., 1989) and
its expression is developm ental^ regulated (Amat e t al.,
1990,1991; Koppel e t al., 1990; Okazaki e t al., 1993).
Sobel and his colleagues proposed th a t this protein might
play a general role as an intracellular signal-relay molecule
integrating diverse signals regulating cellular proliferation and
differentiation (reviewed in Sobel e t al., 1989; Sobel, 1991).
It is observed th a t phosphorylation of stathm in/pl 9 is
rapidly induced and regulated in PCI 2 cells and striatal neurons
by extracellular stimuli - nerve growth factor (NGF) and
vasoactive intestinal peptide (VIP), respectively (Chneiweiss e t
al., 1992; Doye e t al., 1990], indicating the roles stathm in/p1 9
play in neuronal growth an d /o r activation.
In adult mouse brain, where neurons are m ature and
normally active, stathm in/p19 is proportionally much more
phosphorylated than in embryonic brain (Chneiweiss e t al., 1989;
Chneiweiss e t al., 1991; Sobel, 1991), suggesting th a t the
developmental sta te of the cell, as well as its physiological
environment, may synergically determ ine the phosphorylation of
stathm in/p19.
Phosphorylation of sta th m in /p l 9 was observed in many
biological system s in correlation with th e pharmacological
regulations of cell proliferation, differentiation, and functions,
th a t is, pituitary cells and tissue (Sobel e t al., 1983;
Pasmantier e t al., 1986; B eretta e t al., 1988, 1989), muscle
cells (Toutant and Sobel, 1987), HL-60 cells (Feuerstein and
Cooper, 1983; Braverman e t al., 1986) and lymphocytes (Hanash
e t al., 1988; Cooper e t al., 1989; Mary e t al., 1989; Peyron e t al.,
1989), PC12 cells (Doye e t al., 1990), and neurons (Chneiweiss
e t al., 1989).
The expression of stathm in/pl 9 is regulated by numerous
factors, mostly related to the proliferation and differentiation
sta te s of cells.
In the adult rat, stathm in/pl 9 is expressed a t very
different rates in different tissues, both a t the protein and
mRNA levels (Koppel e t al., 1990). It is m ost abundant in brain
(Chneiweiss e t al., 1989; Schubart e t al., 1987, 1988). The
levels of stathm in/pl 9 protein and mRNA in brain reach a peak
around birth (Doye e t al., 1989; Schubart e t al., 1988). The range
of stathm in/pl 9 concentration is 1 to 20 a t the neonatal stage,
brain still being the richest. Compared with the range of the
concentration of 1 to 200 in the adult rat, the high expression of
stath m in /p l 9 and its lower tissue dependence a t th e neonatal
stag e m ost likely reflects a feature common to developing
tissues, i.e., the importance of the regulation of cell
proliferation and differentiation (Koppel e t al., 1990).
Immunocytochemistry dem onstrates th a t essentially all
neurons and glia transiently synthesize this protein during the
course of their differentiation. As both neurons and glia mature,
th e level of immunoreactive stath m in /p 1 9 declines and
eventually becom es undetectable both in fully differentiated
neurons and in astrocytes (Amat e t al., 1991). In adult CNS, high
level expression of stathm in/pl 9 is restricted to regions where
neuronal and glial differentiation is known to persist
(Chneiweiss e t al., 1992).
In situ hybridization experiments of both brain sections
and cultured neurons revealed th a t stath m in/p l 9 transcripts
were detected in m ost neurons in the adult brain (Himi e t al.,
1994). Although the expression of stathm in/pl 9 seem s to be
neuron-specific in the adult rat brain, a recent study by
Schubart and colleagues showed th a t stathm in/p19 is abundant
during development in m ost immature forms of neurons and glia.
Stathm in/p19 mRNA expression was mainly conserved in
neurons with small cell bodies and short processes, such as
granule cells and interneurons in the cortex, striatum and
thalamus (Himi e t al., 1994). In addition, a population of neurons
along the inner edges of the den tate gyrus showed a high
expression of stathm in/p19 mRNA. Stathm in/pl 9 mRNA was not
expressed in neurons of the brain stem (Himi e t al., 1994).
Thus, the expression of stathm in/pl 9 seem s to be
important primarily in neurons, while it may be expressed in
differentiating glia and neurons during developm ent and even in
the adult (Himi e t al., 1994).
The expression of stath m in /p 1 9 seem s to be ubiquitous,
but it is not detected in mouse, rat (Koppel e t al., 1990;
Schubart, 1988) or Xenopus (Maucuer e t al., 1993) liver.
Interestingly, stathm in/p19 gene expression is apparently
repressed in the adult liver, while it is rapidly induced during
liver regeneration (Koppel e t al.. 1990; Okazaki e t al., 1993),
suggesting th at stathm in/pl 9 has a role in cellular
proliferation. The stath m in/pl 9 mRNA upregulation during liver
regeneration supports the hypothesis th a t stathm in/pl 9 is a
growth-associated protein (GAP) (Okazaki e t al., 1993).
Stathm in/pl 9 is also generally well conserved throughout
evolution (Zhu e t al., 1989; Koppel e t al., 1990). It is actually
extremely well-conserved a t least among mammals, the
sequence of the human protein (Zhu e t al., 1989; Maucuer e t al.,
1990) differing by only a single residue from th a t in the rat
(Maucuer e t al., 1990). The coding region of the stathm in/pl 9
gene has also been highly conserved in vertebrate evolution
(Amat e t al., 1991), suggesting th a t stathm in/pl 9 might have an
essential and m ost likely general role in cellular regulatory
processes.
Finally, It has been well noted th a t the sequence of
stathm in/p19 displays a strong homology (Schubart e t al., 1989)
with the neuron-specific developmental protein SCG10 (Stein et
al., 1988), thus indicating th e existence of a corresponding gene
family (Schubart e t al., 1989; Mori e t al., 1990).
Similarities between SCG10 and Stathmin/pl 9
There is a high degree of similarity between SCG10 and
stathm in/pl 9, both a t the level of th e nucleotide sequences of
the corresponding cDNAs and the deduced amino acid sequences
- sharing 69% amino acid identity (Okazaki e t al., 1993; Doye e t
al., 1989; Schubart, 1989; Sobel, 1991).
(1) Genes.
Although SCG10 and stathm in/p19 are encoded by distinct
genes located on different chromosomes (Okazaki e t al., 1993),
they share homologous intron/exon structures. Both genes
consist of five exons, and many of the intron/exon boundaries
fall into the homologous regions of conserved domains of these
two proteins. The coding regions of the both genes have been
highly conserved during mammalian evolution (Schubart e t al.,
1989; Okazaki e t al., 1993).
Thus it can be speculated th a t SCG10 and stathm in/p19
belong to a common gene family (Schubart et al., 1989; Stein et
al., 1988) th a t shares m ost of the stathmin-sequence domain.
Further, the study of the SCG10 gene has indicated th at the
SCG10 gene was diverged from the stathm in/p19 gene by gene
duplication (Okazaki e t al., 1993) i.e., the SCG10 gene arose by
the duplication and divergence of the stathm in/p19 gene and
becam e specialized for functioning in neural tissues (Okazaki e t
al., 1993).
(2) Proteins.
On the other hand, when considering the distribution of
predicted positive and negative charges and th at of polar and
nonpolar residues, the degree of structural resemblance
betw een the two proteins is even greater than suggested by the
degree of sequence identity (Schubart e t al., 1989). Therefore,
it is predicted th at SCG10 and stathm in/p19 closely resemble
each other in secondary structure not only in the two major
regions of strong sequence homology but also in the divergent
sequence th at separates these two regions (Schubart e t al.,
1989).
The structural similarity of the two proteins also includes
three conserved serine residues as potential phosphorylation
sites and a long a-helical domain th a t may serve for coiled-coil
interactions with itself or other cytoskeletal molecules (Sobel,
1991). Since the at least three possible consensus
phosphorylation sites th at the SCG10 amino acid sequence
contains are identical to known phosphorylation sites in
stathm in/p19, SCG10 is thus likely to be a phosphoprotein
(Schubart e t al., 1989; Sobel, 1991).
Furthermore, SCG10 and stathm in/pl 9 share a putative
cytoskeleton-interaction domain in which segmental sequence
similarities were noted with other cytoskeletal molecules such
as myosin, trophomyosin, and cytokeratin (Doye e t al., 1989;
Stein e t al., 1988).
(3) Expression. Functions.
Based on this strong structural resemblance, it is
predicted th at stathm in/p19 and SCG10 have similar functions
(Schubart e t al., 1989).
SCG10 and stathm in/p19 are all expressed in PCI 2 cells
(Schubart e t al., 1989), and both mRNAs are primarily expressed
in neuronal subpopulations but not in glia. The general pattern of
expression of SCG10 is reminiscent of the time course of
stathm in/p19 immunoreactivity in the developing rat brain -
The SCG10 gene expression is maximal in mid to late gestation
of the embryo, and fairly intense in wide areas of neonatal brain
(Mori, 1993). While the abundance of immunoreactive
stath m in /p 1 9 is maximal at birth. They both dramatically
decreases in the a d u lt, but the low levels of their expression
still persist into adulthood (Mori, 1993; Schubart, 1988) -
suggesting th at the expression of both SCG10 and stathm in /pl 9
is developmentally regulated, and they are tw o neuronal
Growth-Associated Proteins (nGAPs) (Amat e t al., 1990, 1991;
Koppel et al., 1990; Mori, 1993; Okazaki e t al., 1993; Schubart e t
al., 1989).
As mentioned above, SCG10 seem s to be a neural-specific
phosphoprotein. Together with the observation th a t SCG10
mRNAs are expressed predominantly in neurons with long
distance-projection axons an d/or extensive dendrites (Himi e t
al., 1994), the available evidence indicates th a t SCG10 may play
a stathmin-like role a t or near nerve terminals and relate to
neuronal plasticity (Himi e t al., 1994).
Another interesting phenomenon is— within SCG10
sequence, A short hydrophobic segm ent is noted to probable
account for its tightly membrane association (Schubart e t al.,
1989), whereas stathm in/pl 9 is a cytosolic protein th a t lacks
such a corresponding hydrophobic region (Schubart, 1982, 1988;
Schubart e t al., 1987; Pasmantier e t al., 1986), however, Stein
e t al (1 9 8 8 ) noted in their studies th at SCG10 can becom e
cleaved in vitro to yield a 19- to 20-kD form th at is no longer
membrane associated.
Distinctions betw een SCG10 and S ta th m in /p 1 9
(1) Neural-Specific
Expression of SCG10 is restricted to the nervous system ,
whereas stathm in/pl 9 is more widely expressed in both
neuronal and non-neuronal tissues
In contrast to neural-restricted SCG10 expression,
stathm in/pl 9 expression is ubiquitous and its protein and
mRNA are found in a wide variety of tissues including the brain,
testis, lung and kidney during development and adult life (Amat
e t al., 1990, 1991; Koppel e t al., 1990; Schubart, 1988; Schubart
e t al., 1989; Sobel, 1991; Sobel, e t al., 1989), but are not
d etected in mouse, rat (Koppel e t al., 1990; Schubart, 198 8 ) or
Xenopus (Maucuer e t al., 1993) liver.
(2) Silencer
The neural-specific expression of the SCG10 gene is
primarily determined by the presence of a neural-restrictive
silencer in the distal region of the SCG10 prom oter (Mori e t al.,
1990, 1992), whereas stathm in/p19 gene lacks such a silencer
elem ent (Schubart e t al., 1989).
Since it has been reasonablly speculated th at the SCG10
gene arose from the stathm in/pl 9 gene by gene duplication and
became specialized for functioning in neural tissues (Okazaki et
al., 1993), it is possible th at th e duplicated copy of the
stathm in/p19 gene acquired this silencer element and th at the
acquisition of this new negative regulatory element restricted
the expression of SCG10 to neurons (Okazaki e t al., 1993).
(3) Membrane-Association
The major structural difference between SCG10 and
stathm in/pl 9, the two homologous proteins, is th at SCG10
contains a stretch of twelve hydrophobic amino acid residues
near the N-terminus th a t is uniquely encoded by the second exon
of the SCG10 gene (Okazaki e t al., 1993), while stathm in/p19
lacks this hydrophobic N-terminal domain (Mori, 1993; Sobel,
1991). In evolution of the SCG10 gene from the previously
existing stathm in/p19 gene, the acquisition of this exon
encoding a hydrophobic peptide seem s to have made SCG10 a
membrane-bound isoform (Okazaki e t al., 1993). The membrane
association property of SCG10 probably reflects a unique role
for this protein in neurons, which m ay not be performed by
stathm in/pl 9 (Himi e t al., 1994).
Thus stathm in/pl 9 is assum ed to be freely diffusible in
the cytoplasm while SCG10 appears to be membrane anchored
(Schubart e t al., 1989).
It is proposed that the exon 2 of the SCG10 gene may be
considered as an addition rather than a duplication, because its
length and sequences are distinct from those of the second exon
of the stathm in/p19 gene (Okazaki e t al., 1993). It seem s that
the acquisition of an exon encoding hydrophobic amino acid
residues presented a novel feature of m em brane association to
the newly emerged copy of the gene family (Okazaki et al.,
1993). This membrane-bound form may permit SCG10 to perform
a stathm in/pl 9-like function in nerve terminals, to which it is
rapidly transported by means of membrane attachm ent.
Alternatively, SCG10 may perform a function a t or near
membranes (Okazaki e t al., 1993).
(4) Length. Promoter Structures
The size of the two genes differ by as much as six times -
the SCG10 transcription unit spans a t least 30 kb, while the
stathm in/pl 9 gene is 6 kb in length (Okazaki e t al., 1993).
The promoter-proximal regions of the SCG10 and
stathm in/pl 9 genes are quite distinct: the SCG10 gene contains
a TATA-box sequence, while the stathm in/p l 9 gene has a GC-
box sequence in addition to a CAT box. This observation
indicates that the two prom oters are not homologous and do not
seem to have arisen by gene duplication, they may have evolved
by fusion of the duplicated coding exons to unique promoters
(Okazaki e t al., 1993).
(5) Genes. Expression
The SCG10 and stathm in/p19 are encoded by distinct genes
located on different chromosomes (Okazaki e t al., 1993).
Southern blot analysis indicates th a t SCG10 mRNA is
encoded by a single gene in the mouse genome, while
stathm in/p19 cDNA probes d e te c t multiple genes (Okazaki e t al.,
1993).
The regional and cellular expression profiles of th e two
related mRNAs were found to be quite distinct (Himi e t al.,
1994).
Stathm in/p19 expression occurs in both neurons and glia
(Amat e t al., 1991; Chneiweiss e t al., 1987), while the
expression of SCG10 is primarily neuron-specific (Anderson and
Axel, 1985; Himi et al., 1994). This su ggests th a t SCG10 and
stathm in/pl 9 have different functions in th e nervous system
(Himi e t al., 1994).
In adult rat brain, SCG10 mRNA is preferentially expressed
in neurons with long-projecting axons an d /o r extensive
dendrites, e.g. mitral cells in th e olfactory bulb, neurons of the
piriform cortex, pyramidal and granule cells of the hippocampus,
Purkinje cells of the cerebellum, and m otor neurons of the brain
stem (Himi e t al., 1994). In contrast, stath m in /p 1 9 mRNA was
weakly distributed over m ost brain regions, and its expression
was mainly conserved in neurons with small cell bodies and
short processes, such as granule cells and interneurons in the
cortex, striatum and thalamus (Himi e t al.,1994).
21
(6) Divergence
Although it is suggested th at in evolution process, SCG10
gene was diverged from the stathm in/p19 gene by gene
duplication (Okazaki e t al., 1993), one more indication on the
distinction betw een these two genes is th at the similarity
between the amino acid sequences of bovine brain stathm in/p19
and rat testis stathm in /p19 is significantly greater than th at
between stath m in/p 19 and SCG10, even when only the
homologous regions of the two proteins are considered.
(Schubart e t al., 1989).
Summary
It could be summarized th at the two proteins are products
of distinct but related genes th a t are members of a common
mammalian gene family (Schubart e t al., 1989; Stein e t al.,
1988) th at shares m ost of th e stathmin-sequence domain.
Proteins like SCG10 are specifically expressed in given
cell types and possess specific additional domains, possibly
evolved by duplication and modification of the more broadly
expressed stathm in/pl 9 gene (Okazaki e t al., 1993).
Stathm in/pl 9, on the other hand, might represent the ubiquitous
and generic functional domain of the protein family, its
regulation and specific functions being related to the particular
sta te of proliferation and differentiation of each cell type
(Sobel, 1991).
The expression profiles indicate th at SCG10 and
stathm in/p19 are two developm ental^ regulated neuronal
Growth-Associated Proteins (nGAPs).
II. Material and Methods
1. Animals and Tissue Sections
Fischer-344 rats of three age groups were used: Young
m ature (6-month-old [n=4]), middle aged (18-m onth old [n=4]),
and aged (24-month-old [n=5)]). No rats showed any gross sign of
pathology at time of use.
The animals were anaesthetized with pentobarbital and
decapitated. Brain samples were immediately removed,
dissected, and frozen a t -20 °C in a dry ice-ethanol bath.
Coronal cryosections were cut into 13urn on a cryostat and
thaw-mounted onto gelatine-coated glass slides. The sections
were stored in a box with desiccant at -80 °C until use.
The in situ hybridizations with SCG10 or stathm in/p19
antisense probes were performed by using adjacent sections
from sam e animals. Each animal has 1 5-20 adjacent sections.
2. In situ Hybridization
The brain sections were warmed to room tem perature for
20 min, then fixed by 4% paraformaldehyde in phosphate
buffered saline (PBS) for 20 min. Slides were rinsed three times
for 10 min each in PBS, once in w ater for 1 min, and once in
0.1 M triethanolamine, pH 8.0 for 1 min. Then the slides were
treated with freshly-prepared 0.25% acetic anhydride/
triethanolamine for 10 min, rinsed for 1 min in 2 x saline-
sodium citrate (SSC), and dehydrated through a series of
upgraded ethanols. Hybridization was performed in a humidified
cham ber for 12-16 h a t 50 °C using 1 x 10 5 cpm per slide of 35S-
labeled RNA probe in 50% formamide, 4 x SSC, 5 x Denhardt’s
solution 1% SDS, 10% dextran sulfate, 0.1 M dithiothreitol, 250
ug/ml of E. coli tRNA, 25 ug/ml of poly (A) and poly (C) under a
cover slip th at was sealed with rubber cement. After
hybridization, the slides were soaked in 4 x SSC with 20 mM
dithiothreitol for 20 min, and rinsed in 4 x SSC for 5-10 min.
The sections were treated with ribonuclease (Boehringer
Mannheim) (20 ug/ml in 0.5 M NaCI, 0.01 M Tris, 1 mM EDTA, pH
8.0) at 37 °C for 30 min, w ashed in 2 x SSC, 20 mM (3-
mercaptoethanol for 2 h a t room tem perature and washed at high
stringency in 0.1 x SSC at 6 0 °C for 1 h. The sections were
dehydrated through a series of upgraded ethanols th at contained
0.3 M ammonium acetate, and air dried. The slides were exposed
to Fuji RX film a t room tem perature, for two to four days.
3. Quantitative Analysis of Film Autoradiography
Film images were captured via lens by an image
processing and analysis system (BQ MEG IV system , Nashville,
TN), then downloaded onto Macintosh Centris 650. The optical
density values were calculated after subtraction of the film
background density by using Mac Software "NIH Image 1.44."
The hybridization signal densities were quantitated over
the following areas: Olfactry bulb, striatum, hippocampus,
midbrain and cerebellum. The nomenclature of the brain regions
are based upon the atlas by Paxinos and W atson (Paxinos and
Watson, 1982), and the quantitation is based upon a series of
known calibration standards. Statistical significance was
assessed by one-way ANOVA, in StatView 5 1 2+. Inter-group
differences were analyzed by post-hoc te s ts (Fisher PLSD &
Scheffe F-test). Results are expressed as m eans ± SEM of the
number of animals. Means were considered to be statistically
significant at p values of < 0.05.
III. Results
SCG10 and Stathm in/pl 9 mRNA expression was studied in
young (6 month-old), middle-aged (18 month-old) and old (24
month-old) rats. In situ hybridization of antisense probes for
th e two mRNAs revealed the facts as follows:
7. Significant Decreases o f S tathm in/p l 9 mRNA Expression in
Hippocampus During Aging
We found th at stath m in/p19 mRNA levels are selectively
reduced in aging rat brain. Representative coronal sections
showing the distribution of the mRNA in young and old rat brains
are shown in Fig.1. It could be observed th a t in the
hippocampus, the stathm in/pl 9 mRNA levels were significantly
lower in old rats (24-month-old) compared with the young rats
(6 month-old). When quantified with image analysis program
(NIH Image 1.44) and compared among the three age groups (6-,
18- and 24-month-old), stath m in/p 19 mRNA levels were
markedly reduced in the aging hippocampus (Fig.3; Table 1). The
age-related reduction appeared to be apparent from middle age.
Comparing middle aged (1 8-month-old) with young (6-m onth-
old) rats, there is 31.5% reduction in CA1, 34.7% in CA2, 38.4%
in CA3 and 27.1% in DG, while comparing old (24-month-old)
with young rats, there is 48.2% reduction in CA1, 44.9% in CA2,
41.6% in CA3 and 38.9% in DG. Overall in hippocampus region,
th e reduction of stathm in/pl 9 mRNA expression level is 44.4%
comparing old with young rats, and 11.3% comparing middle aged
with young rats.
Z. Decrease Trends o f S tathm in/19 mRNA Expression During
Aging in Several Other Regions Examined
Besides the remarkable changes in hippocampus, there are
various other regions examined showing age-related decrease in
stath m in /p 1 9 mRNA expression. With statistically significant
level at p < 0.05 comparing old-aged group and middle-aged
group rats with young-aged group rats respectively, the gene
expression of stathm in/pl 9 in thalamus and hypothalamus
decreases significantly in both age groups (Fig. 1; Fig.3; Fig.4;
Table 1). Meanwhile, several other regions such as cerebral
cortex, piriform cortex, granular layer of cerebellum show
obvious trends of age-related decrease in stathm in/pl 9 mRNA
expression (Fig.1; Fig.3; Fig.4), even though not all the decrease
levels reach statistically significant criteria at p<0.05 (Fig.3;
Fig.4; Table 1).
3. Unchanged Stathm in/p 19 mRNA Expression Levels During
Aging in Olfactory Bulb and other brain regions
In many regions examined including the olfactory bulb,
striatum, midbrain and brain stem , no significant difference
among age groups was found for stathm in/pl 9 mRNA expression.
Quantitation of the hybridization signals confirmed the absence
of age-related changes (Fig 4; Table 1). It indicates th at the
impairment of stathm in/p 19 gene expression in aging rat brain
is spared in these areas.
4. SCG10 mRNA Expression Levels are Primarily unaffected
During Aging in Examined Brain Areas
Interestingly, the age-related gene expression profile of
SCG10, the neural-specific isoform of stathm in/pl 9, was very
different from that of stath m in /p 1 9.
Inspection of sections in which SCG10 mRNA was
visualized by in situ hybridization revealed th at regional
distribution and levels of the hybridization signals in the major
brain regions examined were similar in rats from the various
age groups. Representative coronal sections showing the
distribution of SCG10 mRNA in young (6-month-old) and old (24-
month-old) rat brains are shown in Fig.2. Further, one-way
ANOVA dem onstrated th at primarily there is no statistical
difference among the three age groups for the competition
param eters (p>0.05 for all param eters) in the areas examined
including olfactory bulb, striatum, midbrain, cerebellum and
m ost subregions of hippocampus (Fig.5; Table 2).
In hippocampal subregions examined, the only statistically
significant difference betw een age groups for SCG10 mRNA
levels was in CA2, comparing 24-m onth-old with 6-month-old
rats. However no significant difference was observed in overall
expression levels of SCG10 mRNA in hippocampal formation
between the 6- and 24 month-old rats (Fig.5; Table 2).
29
TABLE 1
QUANTITATIVE ANALYSIS OF THE STATHMIN/P19 mRNA EXPRESSION
LEVELS IN VARIOUS BRAIN REGIONS OF M ALE FISCHER 344 RATS AS DETERM INED
USING IN SITU HYBRIDIZATION HISTOCHEM ISTRY
Y M 0
Olfactory Bulb Section
Glomeruli 23.8 ± 1.90 24.9 ± 2.33 26.4 ± 2.38
Ext. plex 15.1 ± 1.04 15.5 ± 1.42 15.0 ± 1.65
Mitral 35.3 ± 2.59 33.0 ±2.95 32.1 ± 1.59
Int. plex 31.8 ± 5.48 29.9 ± 3.38 28.8 ± 1.93
Granular 37.1 ± 2.47 38.0 ± 2.57 38.9 ± 2.72
OB 27.9 ±4.23 27.5 ± 2.77 28.8 ± 1.93
Striatum Section
CTX 50.1 ± 4 .7 4 44.9 ± 1.89 44.9 ± 2.69
Pir 74.9 ± 1.62 62.1 ± 5.09* 64.0 ± 3.11
Str 21.5 ± 0 .4 9 20.0 ± 1 .5 1 22.2 ± 2.74
Sep 30.8 ± 1 .1 5 27.1 ± 4.42 27.2 ± 2.83
DB 40.1 ± 5.12 37.6 ± 4.28 42.1 ± 6.01
Hippocampus Section
CTX 60.0 ± 3.31 41.5 ± 2 .0 9 * 36.8 ± 1.63*
Pir 68.0 ± 7 .1 0 52.2 ± 2.27 47.5 ± 5.31*
CA1 37.1 ± 4 .8 8 25.4 ± 1.97* 19.2 ± 1.80*
CA2 41.2 ± 7.19 26.9 ± 1.45* 22.7 ± 0.75*
CA3 48.5 ± 6.56 29.9 ± 2.53* 28.3 ± 3.46*
D G 49.9 ± 4.03 36.4 ± 1.44* 30.5 ± 2.52*
HP 32.4 ± 5.88 20.3 ± 1.54* •18.0 ± 1.66*
TH 48.6 ± 5.48 35.4 ± 1.20* 31.1 ± 1.59*
HT 43.9 ± 5.87 31.1 ± 3.36 30.9 ± 3.14
Midbrain Section
CTX 56.1 ± 2.64 50.7 ± 1.14 49.1 ± 2.15
Ent 29.7 ± 1.89 40.9 ± 4 .1 0 30.7 ± 5.45
Colli 37.4 ± 4.94 41.3 ± 3.63 37.1 ± 4.60
Pon 40 .4 ± 6.65 45.2 ± 6.24 37.9 ± 5.48
M id 29 .0 ± 3.46 34.8 ± 3.91 27.4 ± 2.36
Cerebellum Section
Granular 63.6 ± 5.08 56.4 ± 0.97 48.8 ± 3 .8 1 *
Stem 25.3 ± 1.67 26.3 ± 2.64 23.2 ± 1.31
Values represent the mean ± SE for 3-5 animals per age groups.
Y = young (6-mo-old), M = middle-aged (1 8-mo-old), 0 = old (24-mo-old)
* p < 0.05 compared with young rats.
30
TABLE 2
QUANTITATIVE ANALYSIS OF THE SCG10 mRNA EXPRESSION
LEVELS IN VARIOUS BRAIN REGIONS OF M ALE FISCHER 344 RATS ASDETERMINED
USING IN SITU HYBRIDIZATION H ISTOCHEM ISTRY
Y M 0
Olfactory Bulb Section
Glomeruli 12.6 ± 2.34 13.0 ± 3 .3 9 14.6 ± 3.05
Ext. plex 8.3 ± 3.27 8.1 ± 4.14 8.5 ± 3.22
Mitral 44.4 ± 4.41 32.9 ± 5.68 44.2 ± 5.57
Int. plex 26.9 ± 2.42 25.1 ± 3.30 26.1 ± 5.02
Granular 22.3 ± 3.20 20.0 ± 5.47 22.8 ± 3.31
OB 18.2 ± 2.51 17.8 ± 5.20 17.8 ± 2.08
Striatum Section
CTX 31.9 ± 3.68 33.6 ± 2.55 35.6 ± 3.93
Pir 91.6 ± 17.45 95.9 ± 4 .3 2 98.0 ± 5.69
Str 19.7 ± 2.03 20.7 ± 2.26 22.7 ± 3.08
Sep 32.8 ± 3.90 40.7 ± 3.58 42.0 ± 2.78
DB 55.2 ± 10.78 64.7 ± 4.95 52.7 ± 6.34
Hippocampus Section
CTX 33.2 ± 3.02 33.1 ± 3.30 28.5 ± 5.64
Pir 69.2 ± 8.32 77.1 ± 1.79 66.8 ± 13.04
CA1 67.9 ± 6 .1 9 72.8 ± 5.68 68.0 ± 7.76
CA2 95.4 ± 7.10 79.4 ± 5.44 69.5 ± 6.10*
CA3 111.5 ± 7.29 100.0 ± 7.00 97.2 ± 4.81
D G 66.0 ± 6.96 66.4 ± 4.88 65.3 ± 7.02
H P 38.7 ± 4.01 37.0 ± 3.32 34.9 ± 3.36
TH 35.4 ± 4 .1 4 33.7 ± 1.64 33.1 ± 2.74
HT 43.2 ± 6.09 39.4 ± 4.44 40.0 ± 5.17
Midbrain Section
CTX 38.2 ± 5.44 40.1 ± 2.94 37.1 ± 2.78
Ent 38.3 ± 4.84 37.5 ± 1.93 34.8 ± 1.31
Colli 39.3 ± 3.45 38.1 ± 2.68 36.1 ± 2.81
Pon 43.7 ± 5.73 40.8 ± 4.98 44.8 ± 0.88
M id 32.0 ± 4.81 33.0 ± 1.61 34.0 ± 0.73
Cerebellum Section
Granular 125.3 ± 4.72 118.0 ± 8 .4 7 114.9 ± 7.01
Stem 33.5 ± 2.25 26.6 ± 3.87 29.9 ± 4.27
Values represent the mean ± SE for 3-5 animals per age groups.
Y = young (6-mo-old), M = middle-aged (1 8-mo-old), 0 = old (24 -mo-old)
* p < 0.05 compared with young rats.
Fig.1 In Situ Hybridization of Stathm in/p 19 mRNA
in Coronal Sections of Hippocampus of Young and Old
Rat Brains. Computer Processed Film Images of the
Sections Hybridized with 35S-labelled Antisense
Stathm in/p 19 Riboprobe are Shown
Fig. 2 In Situ Hybridization of SCG10 mRNA in
Coronal Sections of hippocampus of Young and Old
Rat Brains. Computer Processed Film Images of the
Sections Hybridized with 35S-labelled Antisense
SCG10 Riboprobe are Shown. The SCG10 Panel
Represents Hybridization Images of the Adjacent
Sections of S tathm in/p19 Panel in Fig.1
Fig.3
120
100
8 0 -
6 0 -
4 0 -
2 0 -
□ 6 mo
H 18 mo
■ 24 mo
CTX Pir CA1 CA2 CA3 DG HP TH HT
The Effects of Aging on Stathm in/pl 9 mRNA
Expression Levels in Hippocampus Section of
Rat Brain
STA T H M IN /P19 mRNA I N MIOBRAIN SECTION ST A T H M IN /P19 mRNA I N OLFA CTO RY S E C T IO N
34
Glomefuli Extptex Mitral Intptex Granular OB
120
100 - « 100
Colli P o n Midi
G ranular Stem
□ 6 m o
□ 1 8 m o
■ 24 m o
Fig.4 The Effects of Aging on Stathm in/pl 9 mRNA
Expression Levels in Olfactory, Striatum,
Mid Brain and Cerebellum Sections of
Rat Brain
SCQtO m R N A I N STRIATUM SECTION
35
□ 6 m o
□ 1 8 m o
■ 24 mo
Glomeruli E xt.piex Mitral Int. plex G ra m ia r OB
Fig.5 The Effects of Aging on SCG10 mRNA
Expression Levels in Different Regions of
Rat Brain
IV. Discussion
These results indicate th at effects of aging on the gene
expression of nGAPs are not a general phenomenon, but rather a
selective one. In the two genes examined, stathm in/p19 mRNA
levels showed significant decrease or trends of decrease in the
aged brain in many areas including the hippocampus, cerebral
cortex, piriform cortex, thalamus, hypothalamus and granular
layer of cerebellum, but not in olfactory bulb, striatum,
midbrain and brain stem . On the other hand, the mRNA levels of
SCG10 generally remained unchanged during aging.
Aging is accompanied by deteriorations of normal brain
function, e.g. learning and memory, which seem s to be caused by
altered neural networks in the aged brain, especially in
hippocampus. The hippocampal neurons display numerous
physiological and structural plasticity which may underlie the
age-related memory deficits (Cotman and Holets, 1985; Cotman
e t al., 1993). Our current results indicate th at an age-related
deregulation of region-specific (or cell-specific) expression of
the stath m in /p 1 9 gene - especially in hippocampus - which may
be influenced by altered transcriptional mechanisms during
aging may be partly responsible for age-related declines in
neuronal and behavioral functions.
While for SCG10, our findings indicate th at declines of
synaptic plasticity in the aging brain are generally not related
to change in th e basal expression levels of SCG10 mRNA.
Although our study fails to support the hypothesis th a t as one of
nGAPs th a t are thought to be critical to the neuronal structural
plasticity, SCG10 was expected th at the general changes in
whose mRNA expression may play a significant role in age-
related changes of neuronal functions (Wang, 1993), it still
remains possible th at SCG10 mRNA levels are selectively
affected in a minor population of neurons which were
undetected in the present study. Furthermore, we need to
emphasize th a t these findings do not rule out age-related
changes in dynamic regulation of mRNA metabolism or changes
at translational or post-translational regulatory steps. The
levels and turnover rates of SCG10 proteins will have to be
measured to assess such changes.
However, one might raise the question as to w hether
SCG10 gene really serves any functional role for the plasticity
of adult central neurons or not. It is possible th at the
persistent, low-level expression of SCG10 mRNA in the adult
brain just simply reflects an incomplete repression of this gene
as a consequence of its initial activation in embryo (Himi e t al.,
1994). According to Cotman and Nieto-Sampedro (19 8 4 ), brain
plasticity can be te ste d in experimental paradigms of lesion-
induced synaptic remodelling. An important phenomenon had
been observed th at following unilateral striatal
deafferentation, SCG10 mRNA was up-regulated in the
contralateral cortex during synaptic remodeling, which suggests
th a t SCG10 plays a fundamental role in the sprouting of
paraterminal axons from the contralateral cortex (Cheng e t al.,
1991; McNeill et al., 1992). Further, it had been found th at the
magnitude of SCG10 mRNA induction was significantly reduced
in aged rats although the time course of th e response was the
sam e as young rats (Cheng e t al., 1993), suggesting th a t the
SCG10 gene does play a functional role in th e adult central
neurons.
To explore why the two highly homologous nGAPs, SCG10
and stathm in/19, have such distinct gene expression profiles in
aging brain, I raise my hypothesis here.
Previous studies have suggested th a t the SCG10 gene
arose by duplication and divergence of th e existing ubiquitously
expressed (neural-enriched) stath m in /p 1 9 gene and became
specialized for functioning in neural tissues (Mori e t al., 1990;
Okazaki et al., 1993). In this reasoning, SCG10 is thought to
arise in the later stage of evolution. It is noted th at the size of
the two genes differ by as much as six times. While the coding
regions of the two genes share high homology, the m ost
prominent distinction betw een the two genes is in introns, as
already characterized in the promoter-proximal regions
(Okazaki e t al., 1993). If we regard SCG10 as having evolved
from duplication and modification of stath m in /p l 9 by acquiring
much longer introns, it is tem pting to speculate th a t the longer
introns in the SCG10 genome contain more transcriptional
regulatory sequences not only for precise temporal and spatial
expression in the nervous system but also functioning as
resisting aging-related reduction of its mRNA expression to
certain degree.
Thus far, the nGAPs whose gene expression has been
examined in aging brain include: SCG10, stath m in /p 1 9, GAP43,
MAP2, a-Tubulin, and synapsin-l, which are all neural-specific
molecules except stathm in/p19 (Mori, 1993; Himi, unpublished).
Since the well developed nervous system arose in much later
stag e of evolution, this group of “neural-specific” molecules
th a t bear certain similar functional roles in neurite outgrowth
may have evolved in relatively later sta g es of evolution and
possess some intriguing common features. In this regard,
SCG10, though has no sequence similarities with other nGAPs
but stathm in/p 19, is probably more close to this group of nGAPs
in functional roles rather than to stathm in/p 19, even though
SCG10 is thought to be m ost close to it in term s of high
sequence homology.
Therefore, it might not be very surprising when the
experiments in our lab dem onstrated th a t among those nGAPs
whose mRNA expression was examined, only the gene expression
levels of stathm in/p 19 were affected significantly by aging
[Himi, unpublished].
On one hand, you can consider th a t the selective reduction
of stathm in/p19 gene expression in aging brain might be caused
by a loss of certain transcriptional factor(s) which is(are)
unique to stathm in/pl 9, and it is of interest to examine DNA-
binding proteins and other transcriptional factors th a t interact
with the stathm in/p 19 gene promoter. On the other hand,
however, we may speculate th a t this “deficient” transcriptional
factor(s) responsible for the aging-related changes of
stathm in/pl 9 gene expression is rather a generic domain which
also exists in SCG10 and other nGAPs mentioned above, while
the stable gene expression levels in aging brain of SCG10 is
achieved by acquiring an additional regulatory element (within
the longer intron) which interacts with (and modifies) th at
generic transcriptional factor(s). This specific domain may
confer a “suppression” of th e selective reduction of mRNA
expression on SCG10 in aging brain, analogous to the “silencer”
function th at has been acquired by SCG10 during evolution to
“suppress” its expression in cells other than neurons.
If this is the possible case, SCG10 appears to be more
intriguing in aging research since its characteristics represent
a group of nGAPs important in neuroplasticity, moreover, since
the comparison with its counterpart stathm in/pl 9 becom es
available - by comparing their prom oter structures. It has
already been indicated th a t th e prom oter structure of the mouse
stathm in/19 gene is distinct from th a t of the SCG10 gene
although the two genes have similar exon/intron organization
and an extensive sequence similarity (Okazaki e t al., 1993).
Thus far, among this group of nGAPs, the m ost well
characterized nGAP is GAP43 (Benowitz e t al., 1990; Fishman,
1989; Gispen e t al., 1992; Pfenninger e t al., 1992; Skene, 1984;
Strittm atter e t al., 1992). Though there is no sequence
similarity betw een SCG10 and GAP43, these two genes share
many common features.
Both GAP-43 and SCG10 are membrane-associated
molecules (Benowitz e t al., 1987; Stein, e t al., 1988). The
variously regulated expression profiles of these two genes are
quite similar, e.g. developmental regulation, NGF-inducibility,
glucocorticoid-suppressibility, upregulation during axonal
regeneration and significant expression in subset neurons of the
adult brain (Anderson and Axel, 1985; Benowitz e t al., 1990;
Fishman, 1989; Gispen e t al., 1992; Himi e t al., 1994; Pfenninger
e t al., 1992; Skene, 1984, 1989; Stein, e t al. 1988; Strittm atter
e t al., 1992).
It was observed th a t in adult brain, even though the SCG10
gene expression was distinct from th a t of GAP-43 (Himi e t al.,
1994) - there is one thing in comm on - they much more
abundantly expressed in regions with higher brain functions, i.e.
evolutionarily later-stage- developed brain regions such as
hippocampus and cerebellum com pared with evolutionarily
early-stage-developed regions like thalamus and hypothalamus,
etc. (Fishman, 1989; Himi e t al., 1994). The density of
stathm in/pl 9 mRNA expression, on the other hand, was observed
more evenly distributed in different se ts of neurons in cerebral
cortex, hippocampus, cerebellum, striatum, thalamus,
hypothalamus and etc. (Himi e t al., 1994).
This observation may further suggest th at among this
group of nGAPs, the neural-specific nGAPs SCG10 and GAP43
have evolved in evolutionary later stages than neural-enriched
nGAP stathm in/p19.
In the brain it has been shown th at mRNA encoding
stathm in/pl 9 was preferentially expressed in short process-
bearing neurons, while SCG10 is more abundantly expressed in
neurons with long processes, large terminal fields, or extensive
dendrites (Himi e t al., 1994). It was suggested th at perhaps the
shorter introns in the stathm in/p19 gene have been optimized
for the regulation of acute response to growth and
differentiation stimuli (Okazaki e t al., 1993b). It is, however,
unknown y et whether this regulation of acute response relates
to certain type of brain function or not.
For the well studied nGAP GAP43 th at is presumed to be a
gene important for long-term structural changes rather than for
immediate responses it has been suggested to be involved in
changes of long-term learning and memory. [Fishman, 1989]
Since an obviously functional deterioration in aging brain
and aging-related degenerative diseases such as Alzheimer's has
been observed th at the short-term memory and acute response
is much more impaired than long-term learning and memory, it
was tempting to speculate w hether long-term memory which
involves more higher brain functions counts more on one
category of molecules such as GAP43 and SCG10, while the
short-term memory, on th e other hand, which involves more
acute response counts more on the other category of molecules
such as stathm in/p19. The unexpected results have indicated
th at the mRNA expression of the molecules presumably
contributing more to "long-term memory" remains relatively
unchanged in aging rat brain. However, there is no ground to
draw any conclusions at this stage. As we know, the functional
deterioration in aging brain involves complex neuronal network.
Many factors synergically contribute to this programmed
process, on transcriptional, translational, and post-
translational levels in various aspects (Finch and Morgan, 1990),
e.g., we can not tell, what is the proportion for individual
molecule contributing to aging-related functional deterioration
in term s of affected basal mRNA expression levels and
decreased ability of gene induction following specific lesion in
the aged brain - the case may vary with each individual
molecule. Furthermore, we need to incorporate these aging
effects with more dynamic mechanisms such as turnover rate,
etc.. Overall, the loss of neuronal plasticity during aging is a
very complex problem in biology of aging, further research is
awaited to reveal more truth about it.
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VI. Abbreviations
Olfactory Section
Striatum Section
Hippocampus Section
Midbrain Section
Glomeruli - glomerular layer olfactory bulb
Ext.plex - ext plexiform layer olfactory bulb
Mitral - mitral cell layer olfactory bulb
Int.plex - int plexiform layer olfactory bulb
Granular - granular cell layer olfactory bulb
OB - main olfactory bulb
CTX - front cerebral cortex
Pir - piriform cortex
Str - striatum
Sep - septofimbrial nu
DB - diagonal band
CTX - mid cerebral cortex
Pir - piriform cortex
CA1-3 - fields CA1 -3 of Ammon's horn
DG - dentate gyrus
HP - hippocampus
TH - thalamus
HT - hypothalamus
CTX - hind cerebral cortex
Ent - entorhinal cortex
Colli - colliculus
Pon - pontine nuclei
Mid - midbrain
Cerebellum Section Granular - granular layer cochlear nuclei
Stem - brain stem
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Creator Cao, Minghua (author)

Core Title Comparison of gene expression of SCG10 and Stathmin/p19 in aging rat brain: an in situ hybridization study

School Leonard Davis School of Gerontology

Degree Master of Science

Degree Program Gerontology

Degree Conferral Date 1995-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,biology, neuroscience,gerontology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor McNeill, Thomas H. (committee chair), Peterson, David B. (committee member)

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA