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Transcript of Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease...
Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
December 29, 1989 Pages 1406-1414
TISSUE-SPECIFIC EXPRESSION OF THREE TYPES OF B-PROTEIN PRECURSOR mRNA:
ENHANCEMENT OF PROTEASE INHIBITOR-HARBORING TYPES IN ALZHEIMER'S DISEASE BRAIP;
Seigo Tanaka'*, Satoshi Shiojiri3, Nobuya Kitaguchi3, Hirataka Ito3,
Yasuyuki Takahashi3,
Jun Kimural, Masakuni Kameyamal,
Shigenobu Nakamura' and Kunihiro Ueda2
1 Department of Neurology, and 2 Department of Clinical Science and Laboratory Medicine, Kyoto University Faculty of Medicine, Kyoto 606, Japan
3 Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co. Ltd., Fuji-shi, Shizuoka 416, Japan
4Department of Neurology, Sumitomo Hospital, Osaka 530, Japan
Received November 21, 1989
Summary Expression of three types of mRNA encoding amyloid B-protein precursor (APP) in various tissues was analysed, using a ribonuclease protection assay, with special reference to Alzheimer's disease (AD). The total content and the proportion of APP mRNAs were specific to each tissue. Among eight tissues examined, the brain was distinct in that the expression level was highest and APP695 mRNA was expressed in abundance. The ratio of APP770/APP751/APP695 mRNAs was approximately 1:10:20 in the cerebral cortex of control brain. The proportions of APP770 mRNA and APP770-plus-APP751 mRNAs increased up to 2.6- and 1.4- fold, respectively, in various regions of AD brain compared with control. The enhanced expression of protease inhibitor-haboring types (APP770 and APP751) may disturb the balance between biosynthesis and degradation of APPs and ultimately lead to accumulation of S-protein as amyloid. D 1989 Academic *lx?*s, Inc.
Deposition of amyloid B-protein in senile plaques and cerebral
vessels is a characteristic finding in the brain of Alzheimer's disease
(AD) (I-5). This protein forms part of a precursor (amyloid B-protein
precursor, APP), as revealed by complementary DNA (cDNA) cloning (6-9).
Thus far, three types of APP mRNA have been found in the human brain
(10,ll); two of them, APP770 and APP751, harbor a protease inhibitor
(10,12,13), while the other, APP695, lacks this inhibitor-(6). These
three APPs are generated from a single transcript by the mechanism of
alternative splicing (IO). Between AD and control, some differences in
expression of total or particular type(s) of APP mRNA have been found in
*To whom correspondence should be addressed.
0006-291W89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved. 1406
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several brain regions by the use of in situ hybridization (14-16) or --
Northern blot analysis (11,171. Our previous study (ll), for example,
showed that the expression of APP770 mRNA increased significantly in
the frontal cortex of AD patient compared with control. In the present
study, we determined, using a ribonuclease protection assay, the
proportion of APP mRNAs in the brain (nine regions) and other nonneural
tissues, with special reference to AD. The results obtained were
indicative of a possible role of the APP-derived protease inhibitor in
amyloidogenesis in the AD brain.
MATERIALS AND METHODS
Preparation of RNA from tissue samples ---___ Postmortem organs were obtained from one AD patient [83 years old
(y.o.)l and five controls (59, 61, 80, 84 and 85 y.0.) without dementia. These organs were removed within 3-l 0 hours frozen at -70°C until use for RNA extraction.
after death and kept
Brains were also obtained from five AD patients and five nondemented controls. One half of the brain was kept frozen, while the other half was fixed in formalin for histological examination. The following nine regions were dissected from each frozen brain; frontal cortex (Brodmann areas 9 and lo), parietal cortex (area 7), temporal cortex (areas 20, 21 and 221, occipital cortex (areas 17, 18 and 191, hippocampus, putamen, thalamus, pons, and cerebellar cortex.
Total cellular RNA was extracted from each tissue by the guanidinium/CsCl method (18).
Ribonuclease protection assay A Dde I (nt 7741-Q I (nt 1136) fragment of -- APP770 cDNA (lo),
which spans over segments 7 and 8 (Fig. l), was reversely linked to a polylinker site (Promega).
downstream to the SP6 promotor in pSP64 plasmid The plasmid DNA thus composed was transcribed with SP6 RNA
polymerase (5 units) in the mixture (10 ul) of 40 mM Tris-Cl (pH 7.5), 6 mM MqC12, 2 mM spermidine, 0.5 mM each ATP, UTP, and GTP, [a-32P]CTP (400 Ci/mmol). The mixture
12.5 uM was incubated at 4O'C for 60
min, and then treated with 1 ug of RNase-free DNase I at 37'C for 10 min. After phenol/chloroform the precipitate
extraction and ethanol precipitation, was dissolved ' hybridization buffer (80%
formamide, 40 mM PIPES (pH 6.4), 4C?On mM NaCl 1 mM EDTAI, and examined for radioactivity by the liquid scintillation method. RNA
-~;7yqo 3"s;-~ayell~y;~~~ed (5i; g5 "c',,f ha~dbri~~~astoilo,n,io~uf~ea~
heated at 85'C for 5 min and incubated at i5'C for 12 hours. To the solution was added 300 ~1 of digestion buffer (10 mM Tris-Cl (pH 7.51, 300 mM NaCl, ribonuclease Tl
5 mM EDTAI containing ribonuclease A (40 u g/ml) and (2 uq/ml) (20,211, and the mixture was incubated at
25OC for 10 min. The mixture was supplemented with 20 ~1 of 10% SDS and 10 ~1 of proteinase K (5 mq/ml), and incubated at 37'C for 15 min, followed by extraction with in ethanol with 10 uq of tRNA
phenol/chloroform and precipitaion as carrier. The pellet was dissolved
inlue, loading buffer [80% formamide, 1 mM EDTA (pH 8.0), 0.1% bromphenol
0.1% xylene cyanol], and RNA hybrids were denatured and fractionated by electrophoresis in 6% polyacrylamide/lO M urea gel. Densitometry of autoradiogram was carried out with Jookoo densitometer (type PAN-802).
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The use of identical amounts of RNA samples enabled us to compare total contents of APP mRNAs among tissues.
Histological estimation of senile _ plaque density Thin sections of the brain (frontal cortex) were examined by a
modification of Bodian stain (22) for abundance of senile plaques under microscope. The average density of plaques was estimated in four arbitrary degrees, - % +++, and designated as SP score.
RESULTS AND DISCUSSION
The strategy of our ribonuclease protection assay is depicted in
Fig. 1. Based on known sequences of APP cDNAs (6,101, 367-, 261-, and
106-nucleotide (nt) bands were interpreted to represent mRNAs encoding
APP770, APP751, and another possible type, APP714, respectively. A
93-nt band was ascribed fully to APP695 mRNA, because the 106-nt band
representing an alternative counterpart, APP7 14 mRNA, was not
detectable under our conditions used (see below).
Expression of APP mRNAs was tissue-specific with respect to both
the total level and the proportion of types. In the brain (frontal
cortex) were expressed three types of mRNA encoding APP770, APP751 and
APP770 mRNA
mRNA:
APP 770
APP 75 1 6 ? 9
6 789
5’++3’ 367 nt
(APP 714)
APP 695 I 6 9
,-Y 93 I 149
\ , ‘--’
FIG. 1 Ribonuclease protection assay of APP mRNAs. Segment Nos. 6, 7, 8 and 9 correspond to the numbers of exons in the APP gene (19); Segment 7 has a protease inhibitor activity. Solid lines with the lengths in nucleotides (nt) represent fragments of the antisense RNA probe protected from ribonuclease digestion, and broken arcs represent regions to be digested. The region encoding B-protein is hatched and marked by "B".
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Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
APP695, as we reported previously (10,ll) (Fig. 2A). Quantification of
autoradiographic bands indicated that the total content of APP mRNAs was
AC AC AC AC AC AC AC AC
367 --- r..-*irr 1Iawc- - -
261 -m-*!r- .“? -.‘..-__
106- 93--w
49-w -
B
Brain
Kidney
Heart
MUSCk
Lung
Proportion (%)
50
mRNA :
Pancreas
Spleen
F
32 / 6?3 APP770
o.ato.4 / 5’2 0 APP751
Liver 0.7Yl.4, 0.7’0.3 0 APP695
FIG. 2 Analysis of APP mRNAs in eight tissues by ribonuclease protection assay. (Cl.
A. Autoradiograms of AD (A) and an example of control 3. Relative proportions of APP mRNAs calculated from band
densities of autoradiograms residues in
with correction for the number of cytonine each fragment. Mean values + S-D. of five controls are
shown, taking the total content in the brain as 100%.
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Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
highest in the brain (Fig. 2B). In other nonneural tissues, APP770
and APP751 mRNAs were also found in varying contents and proportions,
whereas APP695 mRNA was barely detectable (cf. ref. 13, for human fetal
tissues; ref. 23, for mouse tissues). Nowhere was detected APP714 mRNA.
The total content in the kidney was about a half of that in the brain,
and the contents in other tissues were much lower than these two. The
proportion of mRNAs varied markedly among tissues, but hardly among
individuals in each tissue, irrespective of AD or control. In the
brain, the major component was a brain-specific type, APP695 mRNA, while
the minor component, APP770 mRNA, formed the smallest proportion among
the tissues examined.
We, then, examined whether the abundance and proportion of
APP mRNAs observed in the frontal cortex were shared by other regions of
the brain (Fig. 3A). Apparently, the total content of APP mRNAs varied
much less among regions than among tissues, both in AD and control.
APP751 and APP695 mRNAs were dominant throughout the brain. APP770 mRNA
was also found in all regions, but to a lesser extent. APP714
mRNA was undetectable anywhere in the brain. The mean ratio of
APP770/APP751/APP695 mRNAs was approximately 1 : 10*2 : 20 i 4
in cerebral cortices of control brain (Fig. 3B). In contrast,
some difference in band densities was noticed between AD and
control; particularly, two patients [a and b in Fig. 3A) with a
high density of senile plaques upon histological examination
exhibited remarkably denser bands of APP770 mRNA. Quantification by
densitometry indicated that AD brain, as compared with control,
contained a significantly higher proportion of APP770 mRNA in
temporal and occipital cortices, and hippocampus; the content of
APP770-plus-APP751 mRNAs was also high in various regions of cerebral
cortex. APP770 mRNA and APP770-plus-APP751 mRNAs formed higher
proportions in other regions of AD brain, but the difference did not
reach a statistically significant level. The ratio of AD/control in
various brain regions was 1.32 - 2.56 for APP770 mRNA, 0.95 - 1.31 for
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Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Frontal Cx.
A r-+l-=l
abcde fghij
36, ---- .m
261 -0 -*I*
106 - 93 -- )-I-,.- -‘i*
B
Frontal cortex
Parietal cortex
Temporal cortex
Occipital
cortex
Hippocampus
Putamen
Thalamus
Pons
Cerebellum
Temporal Cx.
Flr=l abcde fghi i
.“--
Hippocampus
+-%=-I abcc fghi j
Proportion (%)
50
Cerebellum
acd fghij
AD CTL
AD
CTL
AD CTL
AD CTL
47212
35?7 55t1,
31+9 I 57110 -I
N
5 5
4 5
5 5
5 5
4 5
5 5
5 5
3 5
3 5
mRNA : m APP770 a APP751 El APP695
FIG. 3 Analysis of APP mRNAs in various brain regions by ribonuclease protection assay. A. Autoradiograms of AD and control (CTL) brains in four regions. Samples were obtained from: a, 79 years old (y.o.), SP score (+++); 68 y.0. (+++); c, 87 y.o. (++I; d, 57 y.o. (+); e, 74 y.o. (+); f,b'64 y-0. (-1; g, 74 Y.O. (-); h, 81 y.o. (-); i, 85 y.0. c-j; and j, 94 y.0. (-). E. Proportions of APP mRNAs in nine regions. Mean values + S.D. of the AD group (N = 3-5) and the control (CTL) group (N = 5) are shown. Significance of the difference between AD and CTL was ~~0.05 (p), p<O.Ol (P*), or pcO.001 (*+*) (Student t- test). The.significance of difference in APP770-plus-APP751 mRNAs is identical to that of APP695 mRNA.
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Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
APP751 mRNA, 0.81-O-98 for APP695 mRNA, and 1.05 - 1.41 for APP770-
plus-APP751 mRNAs.
The mechanism of amyloidogenesis and the reason for preferential
involvement of the brain are not known. Our present study unveiled
several unique features of APP mRNA expression in the brain: (i) the
highest total content of APP mRNAs; (ii) a specific expression of
APP695 mRNA, i.e., a protease inhibitor-lacking type, and (iii) higher
proportions of APP770 and APP751 mRNAs, i.e., protease inhibitor-
horboring types, in AD brain, especially in cerebral cortices where the
density of amyloid is highest (24). These findings, altogether, appear
to support the view that the balance between biosynthesis and
degradation of APPs in the brain is maintained by the proportion of
inhibitor-horboring to inhibitor-lacking APPs, and that an increase in
the former types may disturb the balance and eventually lead to
accumulation of aberrant metabolite(s), such as B-protein. A
possibility of the existence of brain-specific protease engaged in
APP degradation and suppressed by the APP-derived inhibitor remains to
be investigated.
A marked difference in proportions of APP mRNAs among tissues is
indicative of a mechanism that regulates tissue-specific splicing
of the gene transcript. Whether the alteration of APP mRNA proportion
in AD brain is due to a change in splicing or in cell types, such as
gliosis, is currently under investigation. In this context, a recent
report is noteworthy that an immunoreactivity to B-protein was found in
nonneural tissues, including the skin, subcutaneous tissues and the
intestine, of AD patients (25). Analysis of APP mRNAs in these tissues
would be challenging for our hypothesis of the mechanism of
amyloidogenesis.
ACKNOWLEDGMENTS
Gratitude is extended to Drs. T. Takeda, I. Saito, W. Araki (Kyoto University) and R. Kodaira (Asahi Chemical Industry Co. Ltd.) for useful discussion. We are also grateful to Drs. M. Ogawa, F. Udaka, K. Hara and R. Matsumoto (Kyoto University-affiliated hospitals) for providing us with tissue samples, and Dr. S. G. Younkin (Case Western
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Reserve University) for informing us of their paper in press. This work was partly supported by Grants-in-Aid and Special Grant for Clinical Investigation from the Ministry of Education, Science and Culture, Japan, and a grant from Sasagawa Health Science Foundation.
Note Added in Proof During the preparation of this manuscript, we were informed that
Golde et al __ __. (26) had prepared a report on analysis of APP mRNA expression and detection of APP714 mRNA at a low level in the brain and other tissues by the use of polymerase chain reaction.
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