Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease inhibitor-harboring types in Alzheimer's disease brain

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


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).

1407


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".

1408


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%.

1409


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

1410


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.

1411


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

1412


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.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

REFERENCES

Mountjoy, C.Q., Tomlinson, B.E., and Gibson, P.H. (1982) J. Neurol. Sci. 57, 89-103. Glenner, G.G., and Wong, C.W. (1984) Biochem. Biophys. Res. Commun. 120, 885-890. Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. USA 82, 4245-4249. Wong, C.W., Quaranta, V., and Glenner, G.G. (1985) Proc. Natl. Acad. Sci. USA 82, 8729-8732. Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.N., and Beyreuther, K. (1985) EMBO J. 4, 2757-2763. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K., and Miiller- Hill, B. (1987) Nature (London) 325, 733-736. Robakis, N.K., Ramakrishna, N., Wolfe, G., and Wisniewski, H.M. (1987) Proc. Natl. Acad. Sci. USA 84, 4190-4194. Goldgaber, D., Lerman, M-I., McBride, O.W., Saffiotti, U., and Gajdusek, D.C. (1987) Science 235, 877-880. Tanzi, R-E., Gusella, J.F., Watkins, P.C., Bruns, G.A.P., St George-Hyslop, P., Van Keuren, M.L., Patterson, D., Pagan, S., Kurnit, D.M., and Neve, R.L. (1987) Science 235, 880-884. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S., and Ito, H. (1988) Nature (London) 331, 530-532. Tanaka, S., Nakamura, S., Ueda, K., Kameyama, M., Shiojiri, S., Takahashi, Y., Kitaguchi, N., and Ito, H. (1988) Biochem. Biophys. Res. Commun. 157, 472-479. Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., HSU, D -I Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F ., and Cordell, B. (1988) Nature (London) 331, 525-527. Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J-F., and Neve, R.L. (1988) Nature (London) 331, 528-530. Cohen, M.L., Golde, T.E., Usiak, M.F., Younkin, L.H., and Younkin, S.G. (1988) Proc. Natl. Acad. Sci. USA 85, 1227-1231. Higgins, G.A., Lewis, D.A., Bahmanyar, S., Goldgaber, D., Gajdusek, D.C., Young, W-G., Morrison, J.H., and Wilson, M.C. (1988) Proc. Natl. Acad. Sci. USA 85, 1297-1301. Palmert, M.R., Golde, T-E., Cohen, M.L., Kovacs, D.M., Tanzi, R-E., Gusella, J.F., Usiak, M.F., Younkin, L.H., and Younkin, S.G. (1988) Science 241, 1080-I 084. Johnson, S.A., Pasinetti, G.M., May, P.C., Ponte, P.A., Cordell, B, and Finch, C.E. (1988) Exp. Neurol. 102, 264-268. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 5294-5299. Lemaire, H.G., Salbaum, J-M., Multhaup, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K., and Mtiller-Hill, B. (1989) Nucl. Acids Res. 17, 517-522. Melton, D.A., Krieg, P.A., Rebagliati, M-R., Maniatis, T., Zinn, K ., and Green, M.R. (1984) Nucl. Acids Res. 12, 7035-7058.

1413


21. Gilman, M. (1987) In Current Protocols in Molecular Biology (Ausubel, F. M. et al., Eds) 4.7.1-4.7.8 John Wiley & Sons, New York.

22. Bodian. D. (1936) Anat. Rec. 65, 89-97. 23. Yamada, T., Sasaki, H, Dohura, K., Goto, I., and Sakaki, Y. (1989)

Biochem. Biophys.Res. Commun. 158, 906-912. 24. Ogomori, K., Kitamoto, T., Tateishi, J., Sato, Y., Suetsugu, M.,

and Abe, M. (1989) Am. J. Pathol. 134, 243- 251. 25. Joachim, C. L., Mori, H., and Selkoe, D. J. (1989) Nature (London)

341, 226-230. 26. Golde, T.E., Estus, S., Usiak, M., Younkin, L.H., and Younkin,

S.G. (1989) Neuron (in press).

1414

Documents

Tissue-specific expression of three types of β-protein precursor mRNA: Enhancement of protease inhibitor-harboring types in Alzheimer's disease brain