Vol. 157, No. 2,1988

December 15,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 472-479

THREE TYPES OF AMYLOID PROTEIN PRECURSOR mRNA IN HUMAN BRAIN:

THEIR DIFFERENTIAL EXPRESSION IN ALZHEIMER'S DISEASE

Seigo Tanaka I*, Shigenobu Nakamura I , Kunihiro Ueda 2, Masakuni Kameyama 3, Satoshi Shiojiri 4, Yasuyuki Takahashi 4,

Nobuya Kitaguchi 4 and Hirataka Ito 4

IDepartment of Neurology and 2Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University,

Sakyo-ku, Kyoto 606, Japan

3Department of Neurology, Sumitomo Hospital, Osaka 530, Japan

4Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co. Ltd.,

Fuji-shi, Shizuoka 416, Japan

Received October 12, 1988

Summary Three types of amyloid protein precursor (APP) mRNA, produced by alternative splicing, were detected by Northern blotting in human brains, both control and Alzheimer's disease. These mRNAs encode APP695 consisting of 695 amino acids, APP751 harboring a 56 amino acid insert homologous to a Kunitz-type trypsin inhibitor inside APP695, and APP770 containing an additional 19 amino acid insert. Another possible APP mRNA which encodes "APP714" containing a 19 amino acid insert was not found in brain samples tested. Quantitative analysis revealed that, although the relative expression levels of the three mRNAs were variable among individuals, there was no remarkable change in expression of APP695 and APP751 mRNAs in Alzheimer's disease compared with control, but that APP770 mRNA level was elevated significantly in Alzheimer's disease. ©1988AcademicPress, Inc.

A major neuropathological finding of Alzheimer's disease is

deposition of amyloid 8-protein in senile plaques and cerebral

vessels (I-5). Complementary DNA (cDNA) clones encoding the amyloid

protein precursor (APP) have been isolated and sequenced (6-9). This

precursor protein (APP695), comprising 695 amino acids as deduced

from the base sequence, has structural features characteristic of

cell surface receptors with a large extracellular hydrophilic domain

*To whom correspondence should be addressed.

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 472

Vol. 157, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and a membrane-spanning hydrophobic region near the carboxyl terminus.

Recently, the core protein of heparan sulfate proteoglycan was found

to be homologous and antigenically related to APP (10), which is

indicative of their possible identity.

From a cDNA library of a human glioblastoma cell line, we

isolated APP cDNA clones (11), and found two new species (pAPP751 and

pAPP770), together with a cDNA clone (pAPP695) coding for a protein

apparently identical to that previously reported by others (6-9). The

new clones harboring the sequence for APP751 and APP770 have a 168-bp

insert (11-13) and a 225-bp insert (11), respectively, inside the

coding region of pAPP695. Cloning of genomic DNA from a human

leukocyte DNA library has revealed that the 225-bp insert in pAPP770

is derived from two exons, 168-bp and 57-bp long, that are separated

by an intron of about 3 kb long, and that the former exon corresponds

exactly to the 168-bp insert in pAPP751. These two exons and their

flanking ones are tentatively designated as H, I, J and K in the order

of 5' to 3' direction (Fig. I). As judged by the result of Southern

blot analysis, the 225-bp insert (I-J) exists as a single copy per

haploid of human genome, suggesting that the three species of APP mRNA

are produced by alternative splicing (11).

It is of interest that the sequence of 56 amino acids (fragment

I) encoded by exon I shows a close similarity to the basic trypsin

inhibitor family (Kunitz type) (11-13). In fact, we observed a higher

activity to inhibit trypsin in the lysate of COS-I cells

transfected with pAPP770 compared with pAPP695-transfected cells

(11). It seems possible that inhibition of protease(s) by APP751

and/or APP770 might cause aberrant catabolism of APP and lead to

accumulation of amyloid 8-protein.

In view of the fact that the expression of exon I and J in adult

human brains has not been studied in detail, we undertook Northern

blot analysis of APP mRNAs obtained from the brains of Alzheimer's

473

Vol. 157, No. 2, 1 9 8 8 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

disease patients and controls using site-specific oligonucleotide

probes.

MATERIALS AND METHODS

Postmortem brains were obtained from nine Alzheimer's diseas~ patients and eight age-matched controls. Diagnosis was confirmed b] histological examinations. The brains were removed within 3-10 hour~ after death, and kept frozen at -70°C until use. Total cellular RNJ was prepared from frontal cortex (Brodmann areas 9 and 10) of eact brain by the guanidium/CsCl method (14), and poly(A)+RNA wa~ isolated by oligo(dT)-cellulose chromatography (15). By criterion oI intactness of 8-actin mRNA, three samples of Alzheimer's disease an6 four controls were selected for analysis of APP mRNAs.

Northern blot analysis was performed using syntheti( oligonucleotide probes, AM-11, AM-13, AM-14 and AM-15, designed t( differentiate four possible APP mRNA species (Fig. I) under the stringent hybridization conditions employed (11). Glyoxal-denaturec poly(A)+RNA (4.0 pg per lane) was resolved by electrophoresis in 1.0~ agarose gel, and transferred to a Zeta Probe filter (Bio-Rad) (16) The filter was subjected to hybridization with one of the 32p-labellec oligonucleotide probes in 5 x SSC, containing 25 mM sodium phosphat~ (pH 7.0), 5 x Denhardt's solution, I% glycine, and 0.1% SDS at 55°( for 2 hours, and washed at 55°C in 6 x SSC containing 0.1% SD~

p A P P 6 9 5

p A P P 6 9 5

pAPP751 l/

pAPP770 l/

(PAPP714) l/

8om H I 8om HI Bgl I I

/ . . - '~,ml AM-1 1

. !/ I~[ ,,AM-13

I k , I •

-'"'-. i I~ AM-14

A M - 1 5 Iml ~ A M - 1 4

8"

Fiqure [. Schematic representations of APP cDNAs and synthetic oligonucleotide probes. The putative coding region of APP cDNA is shown by open boxes, and that of 8-protein by a solid box as marked by an arrowhead. I and J stand for inserts, and H and K for the adjacent exons (11). Four oligonucleotide probes and their hybridization sites (indicated by bars) are as follows:

AM-11 (5'-CTGTTGTAGGAACTCGAACCACCT-3') for the H-K junction, AM-13 (5'-CTGTTGTAGGAATGGCGCTGCCAC-3') for the I-K junction, AM-14 (5'-CTGTTGTAGGAAGTTTAACAGGAT-3') for the J-K junction, AM-15 (5'-AAACTTTGGGACACTCGAACCACCTC-3') for the H-J junction.

474

Vol. 157, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(11). After autoradiography, the probe was washed out, and the filter reused for hybridization with another probe. Densitometry of autoradiograms was carried out with Jookoo PAN-802 type densitometer.

RESULTS AND DISCUSSION

Hybridization with three oligonucleotide probes, AM-t1, AM-13 and

AM-14, revealed specifically the presence of respective 3.2-3.4 kb

mRNAs (Fig. 2). This size coincided with those of APP mRNAs reported

previously (6-9). Although there was a considerable variation in the

expression level among individuals in both control and Alzheimer's

disease groups, these results presented unequivocal evidence that both

APP751 mRNA (H-I-K type) and APP770 mRNA (H-I-J-K type) were expressed

in the human brain. Another possible APP ("APP714") mRNA (H-J-K type)

was not detectable with AM-15 probe under our conditions used. The

bands given by AM-14 probe, therefore, was considered to represent

entirely APP770 mRNA.

For a quantitative comparison of expression levels, the relative

density, i.e. the ratio of the density of APP band to that of

A M - 1 1

1 2 3 4 5 6 7 k b

iiii:~iii :~t:ii~{iii:::iii~:~"i~"iii~,i{iiiiii~iii!!ii~i4~iiii; ~';'~i:~ii#ii`iiii; - - 4 . 4

,"~i "iii!~ , . . ~ i , ~ l

.,L: Itt.l|lli, i~:" :::::,;:;::. ::li!:~m::::z:: :::;~!:;:::ilili fihir ~ . ; i ~ i i i i i i - ; i ~ ! : : .: "iii;iiil!:ii~!~ii:~= "~:" " "i~iW :i ~ " :~i - - 4 • 4

A M - 1 3 ~-' '-'~ i

~i" i~*[ti:: g ;u :: :~: z: : ; ; ;i~ ," ,':-" ,'iiii~!id.'i,':lii~iil ~d i i ! ~ i i ii, i i ~ i ~ i r ~ fi ~ u i : ¢ i : l = l t i l i l i ' . l ~ i f i l . ' ~ l i l . ~ l i u l l i l l ~ i i . ' ~ t ~ l l i 4.4

AM - 1 4 2 , 4

i~.:[~Liil]iiiiiiillii~iiii~Fi'u;iii|~ii~ii~iii'i!~i:;; ;;il.":~iiii" ~ ;!i ";'~i!:'" ; t . r

Fiqure 2. Northern blot analysis of poly(A)+RNA prepared from human brains. Lanes I-4, controls (90, 69, 78 and 57 years of age; diagnosed as multi-infarct dementia, Parkinson's disease, cerebral infarction and multiple sclerosis, in order); lanes 5-7, Alzheimer's disease patients (57, 69 and 87 years of age, in order). Four kinds of hybri- dization probes (Fig. I ) were used as indicated. No band was detected by AM-I 5 probe.

4 7 5

Vol. 157, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8-actin band (Fig. 2) was estimated, and the mean values were

compared between the Alzheimer's disease group and the control group

(Fig. 3). The ratio of the mean value for Alzheimer's disease to that

for control was 1.12 for APP695 mRNA probed by AM-11, and 1.11 for

APP751 mRNA probed by AM-13, indicating no remarkable change in the

expression levels of these mRNAs. By contrast, the ratio for APP770

mRNA probed by AM-14 was 2.04, and the difference was statistically

significant at a level of p<0.05 by Student t-test. Based on these

results, we tentatively concluded that APP770 mRNA expression

specifically increased in Alzheimer's disease. This conclusion

remains to be substantiated by analysis of more cases.

As for the expression of APP mRNAs in Alzheimer's disease,

there has been a controversy among reports. Duplication of amyloid

8-protein gene in Alzheimer's disease was reported by Delabar et al.

(17), who thereby proposed an idea of possible overproduction of

APP mRNA in the patient's brain. This idea, however, has not

necessarily been supported by others (18-21). Rather, Northern blot

analysis indicated that the overall level of APP mRNA in cerebral

cortex was not typically higher in Alzheimer's disease than in

controls (22). On the other hand, in situ hybridization studies

suggested an increase of APP mRNA in neurons of the nucleus basalis

(23), and the differential APP mRNA expression within hippocampal

neuronal subpopulations in Alzheimer's disease (24). In none of

these reports were studied the expression patterns of various APP mRNA

species. Only Palmert et al. (25) reported elevation of the level of

the APP mRNA lacking insert(s) in neurons of the nucleus basalis and

the locus ceruleus in Alzheimer's disease, albeit with no distinction

between APP751 mRNA and APP770 mRNA.

Our preliminary analysis by Northern blotting with

BamHI-BglII fragment of APP695 cDNA (Fig. I) has suggested

476

a shorter

that the

Vol. 157, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A M - 1 1 A M - 1 3 A M - 1 4

2.5

>' 2 .0

r-- o~ "o 1.5

o)

0.5

2.0

"6 1.5

* 3 e4

~ 1.0

e l 0 .5

0 I 0 CTL A 0

2 .5

2 .0

1 .5 e 6

e 4

l O

e l

0 . 5

I 0 CTL A D

2.5 e6

mllm 7

* 4 e5

e l m

* 2 * 3

I C T L A D

Fiqure 3. Relative densities of autoradiograms of Northern blotting analysis for control (CTL) and Alzheimer's disease (AD) groups. The mean value of each CTL group is taken as unity. The numbers beside dots represent the case numbers for which the densities were calculated (Fig. 2). Bars indicate the mean values of relative densities in each group. * p<0.05, Student t-test.

total amount of APP mRNAs does not change appreciably in Alzheimer's

disease (unpublished data). This result, together with the finding of

a significant increase in APP770 mRNA expression unaccompanied by a

change in APP695 or APP751 mRNA in Alzheimer's disease, suggests that

the deposition of amyloid 8-protein is not ascribable simply to

overexpression of APP gene nor to aberrant expression of exon I

coding for a protease inhibitor. Rather, it seems possible that the

coexpression of exon J along with exon I is related to pathogenesis

of Alzheimer's disease. The 19 amino acid peptide (fragment J) encoded

by exon J is not homologous to any of known proteins, and its

physiological function remains to be clarified. An attractive

hypothesis at this moment is that the fragment J affects the protease

inhibitor activity of fragment I, or that the flanking by fragment

J, with or without possible O-glycosylation at a threonine residue of

the Thr-X-X-Pro sequence (11, 26), imposes a conformational change on

APP770, thereby leading to aberrant metabolism of APP and deposition

of 8-protein.

477

Vol. 157, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS

We thank Drs. M. Ogawa, T. Seko, K. Hara, R. Matsumoto and T. Kimura for providing us with brain samples, and Dr. I. Saito and Mr. S. Horiguchi for useful discussion. The work was partly supported by Grants-in-Aid and Special Grant for Clinical Investigation from the Ministry of Education, Science and Culture, Japan.

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