Three types of amyloid protein precursor mRNA in human brain: Their differential expression in Alzheimer's disease

Vol. 157, No. 2,1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 472-479

December 15,1988

THREE TYPES OF AMYLOID

PROTEIN

THEIR DIFFERENTIAL

PRECURSOR

EXPRESSION

mRNA IN HUMAN BRAIN:

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,

4Bio-Science

Sumitomo

Hospital,

Osaka

Laboratory, Life Science Research Asahi Chemical Industry Co. Ltd., Fuji-shi, Shizuoka 416, Japan

530, Japan

Laboratories,

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, b o t h c o n t r o l and A l z h e i m e r ' 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 e x p r e s s i o n of A P P 6 9 5 and APP751 m R N A s in A l z h e i m e r ' s d i s e a s e c o m p a r e d w i t h control, but that A P P 7 7 0 m R N A level w a s e l e v a t e d significantly in Alzheimer's disease. © 1 9 8 8 A c a d e m i c P r e s s , Inc.

A

major

deposition

neuropathological

of

amyloid

vessels

(I-5).

protein

precursor

precursor from

the base

cell

surface

8-protein

Complementary

protein

finding

DNA

in

of A l z h e i m e r ' s

senile

(cDNA) clones

(APP) have been isolated (APP695),

sequence, receptors

*To whom correspondence

comprising has

with

a

structural large

472

encoding

and sequenced 695

amino

features

extracellular

should be addressed.

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

plaques

acids

disease

and

is

cerebral

the amyloid (6-9).

This

as deduced

characteristic hydrophilic

of

domain

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and a m e m b r a n e - s p a n n i n g Recently,

hydrophobic region near the carboxyl terminus.

the core protein of heparan sulfate p r o t e o g l y c a n

to be h o m o l o g o u s

and a n t i g e n i c a l l y

indicative of their possible From

a

cDNA

library

isolated APP cDNA clones

related

to A P P

(10),

of

a

human

pAPP770),

together

apparently

identical to that previously

glioblastoma

cell

a cDNA clone

(11-13)

coding

region

leukocyte

and of

DNA

a 225-bp insert

pAPP695.

library

has

is

line,

we

(pAPP751

and

reported by others and APP770

(6-9).

of

that

genomic the

DNA

225-bp

The

have a 168-bp

(11), r e s p e c t i v e l y ,

Cloning revealed

species

(pAPP695) coding for a protein

new clones harboring the sequence for APP751 insert

which

identity.

(11), and found two new

with

was found

inside

from

insert

the

a human

in pAPP770

is d e r i v e d f r o m t w o exons, 1 6 8 - b p and 5 7 - b p long, that are s e p a r a t e d by an intron of about 3 kb long, exactly

to the

168-bp

insert

and that the f o r m e r

in p A P P 7 5 1 .

These

exon corresponds

two exons

and t h e i r

flanking ones are tentatively designated as H, I, J and K in the order of 5' to 3' d i r e c t i o n blot analysis,

(Fig. I).

the 2 2 5 - b p

haploid of h u m a n genome, are produced

As j u d g e d

insert

by

the r e s u l t of S o u t h e r n

(I-J) e x i s t s

as a s i n g l e

suggesting that the three species of APP m R N A

by alternative

splicing

(11).

It is of i n t e r e s t that the s e q u e n c e of 56 a m i n o I) encoded by exon I inhibitor activity

family to

transfected (11).

It

and/or

shows

(11-13).

trypsin

with pAPP770

seems

a close similarity

(Kunitz type)

inhibit

c o p y per

possible

in

compared that

APP770 might cause

In fact, the

with

inhibition

acids

(fragment

to

the basic trypsin

we

observed

lysate

COS-I

cells

pAPP695-transfected

cells

of

aberrant c a t a b o l i s m

of

a higher

protease(s) of

APP

by

and

APP751 lead to

a c c u m u l a t i o n of a m y l o i d 8-protein. In v i e w of the fact that the e x p r e s s i o n human

brains

blot analysis

has not b e e n s t u d i e d of APP m R N A s

in d e t a i l ,

obtained

473

of e x o n I and J in a d u l t we u n d e r t o o k

Northern

f r o m the b r a i n s of A l z h e i m e r ' s

Vol. 157, No. 2, 1 9 8 8 disease

patients

and

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS controls

using

site-specific

oligonucleotide

probes.

MATERIALS

AND METHODS

Postmortem b r a i n s w e r e o b t a i n e d f r o m n i n e A l z h e i m e r ' s diseas~ patients and eight a g e - m a t c h e d controls. Diagnosis was c o n f i r m e d b] histological examinations. The brains were r e m o v e d w i t h i n 3-10 hour~ after death, and kept frozen at - 7 0 ° C until use. Total cellular RNJ was prepared from frontal cortex (Brodmann areas 9 and 10) of eact b r a i n by the guanidium/CsCl method (14), a n d poly(A)+RNA wa~ isolated by oligo(dT)-cellulose chromatography (15). By criterion oI intactness of 8-actin mRNA, three samples of Alzheimer's d i s e a s e an6 four controls were selected for analysis of APP mRNAs. Northern blot analysis was performed using syntheti( oligonucleotide p r o b e s , A M - 1 1 , A M - 1 3 , A M - 1 4 a n d A M - 1 5 , d e s i g n e d t( differentiate four possible APP m R N A species (Fig. I) under the stringent h y b r i d i z a t i o n conditions e m p l o y e d (11). Glyoxal-denaturec poly(A)+RNA (4.0 p g 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 h y b r i d i z a t i o n w i t h one of the 32p-labellec o l i g o n u c l e o t i d e probes in 5 x SSC, containing 25 m M s o d i u m phosphat~ (pH 7.0), 5 x D e n h a r d t ' s s o l u t i o n , I% g l y c i n e , and 0.1% SDS at 55°( for 2 hours, and washed at 5 5 ° C in 6 x S S C c o n t a i n i n g 0.1% SD~

8om H I

8om HI

BglII

pAPP695

8"

pAPP695 /

pAPP751 l/

..-'~,ml A M - 1 1

.

!/ I~[,,AM-13 I I

k,

•

pAPP770 l/ -'"'-.

i I~ AM-14

(PAPP714) l/ AM-15

Iml ~

AM-14

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). A f t e r a u t o r a d i o g r a p h y , the probe was washed out, and the f i l t e r r e u s e d for h y b r i d i z a t i o n w i t h a n o t h e r probe. D e n s i t o m e t r y of a u t o r a d i o g r a m s w a s c a r r i e d out w i t h J o o k o o P A N - 8 0 2 type d e n s i t o m e t e r .

RESULTS Hybridization AM-14,

revealed

mRNAs

(Fig.

2).

This

(6-9).

expression

level

groups,

APP751

mRNA

in the

human

was

among these

brain.

For density,

by

APP770

results

with

was

probe therefore,

and

type)

were

mRNA

and kb

reported in the

Alzheimer's

evidence

("APP714")

under

3.2-3.4

variation

control

(H-I-J-K

APP

AM-13

of A P P m R N A s

unequivocal

mRNA

possible

probe,

respective

those

both

presented

AM-15

of

AM-t1,

a considerable in

and A P P 7 7 0

with

that b o t h expressed

(H-J-K

type)

our conditions

used.

considered

represent

was

to

The

mRNA.

a quantitative i.e.

there

probes,

presence

coincided

Another

AM-14

the

individuals

(H-I-K type)

given

entirely

size

Although

not detectable

bands

three o l i g o n u c l e o t i d e

specifically

previously

disease

with

AND DISCUSSION

the

comparison

ratio

of

the

1

of e x p r e s s i o n

density

2

3

4

of

5

6

levels,

APP

7

band

the relative to

that

kb

iiii:~iii:~t:ii~{iii:::iii~:~"i~"iii~,i{iiiiii~iii!!ii~i4~iiii; ~';'~i:~ii#ii`iiii; -- 4.4 AM-11

,"~i

,.. ~ i , ~

"iii!~

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 - -

AM - 13

~-'

'-'~

4

• 4

i

~i"i~*[ti:: g;u :: :~: z::;; ;i~ ,",':-",'iiii~!id.'i,':lii~iil ~dii ! ~ i i ii,i i ~ i ~ i ui:¢i:l=ltilili'.l~ifil.'~lil.~liullill~ii.'~t~ll

r ~ fi ~

AM -14

i

4.4

2,4 i~.:[~Liil]iiiiiiillii~iiii~Fi'u;iii|~ii~ii~iii'i!~i:;; t .r

;;il.":~iiii" ~ ;!i ";'~i!:'" ;

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 hybridization probes (Fig. I ) were used as indicated. No band was detected by AM-I 5 probe. 475

of

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8 - a c t i n b a n d (Fig. 2)

was

estimated,

and

the m e a n v a l u e s

c o m p a r e d b e t w e e n the Alzheimer's disease group and (Fig.

3).

was

mRNA

1.12 for A P P 6 9 5

probed by AM-13,

expression mRNA

levels

significant results,

mRNA

was

2.04,

tentatively

specifically

in

by S t u d e n t that

Alzheimer's

was

t-test.

APP770

for

statistically

B a s e d on t h e s e

mRNA

disease.

expression

This

conclusion

to be substantiated by analysis of more cases.

the e x p r e s s i o n

of A P P

mRNAs

in A l z h e i m e r ' s

there has been

a controversy

8-protein gene

in Alzheimer's disease was reported

who

APP mRNA

thereby

analysis cortex

been

proposed

not

(22).

neuronal

higher

in

subpopulations

in

overproduction however,

Rather,

Alzheimer's

of

has not

Northern

level of A P P m R N A

in situ

APP m R N A

by Delabar et al.

possible

(18-21).

the other hand,

the differential

of

disease,

Duplication of a m y l o i d

This idea,

the o v e r a l l

typically

suggested an increase of and

idea

by others

that

On

an brain.

supported

indicated

was

controls

among reports.

in the p a t i e n t ' s

necessarily

(23),

and 1.11

the ratio for APP770

and the difference

concluded

increased

As for

(17),

group

no r e m a r k a b l e change in the

By contrast,

at a l e v e l of p<0.05

we

p r o b e d by AM-11,

indicating

of these mRNAs.

probed by AM-14

remains

the control

The ratio of the m e a n value for Alzheimer's disease to that

for c o n t r o l APP751

were

blot

in c e r e b r a l

disease

than

hybridization

in

studies

in neurons of the nucleus basalis

APP m R N A

expression

Alzheimer's

disease

within

hippocampal

(24).

In

none

of

these reports were studied the expression patterns of various APP mRNA species.

Only Palmert

the APP m R N A

(25)

lacking insert(s)

the locus ceruleus b e t w e e n APP751

reported

elevation

of the

level of

in neurons of the nucleus basalis and

in Alzheimer's disease,

albeit with no distinction

m R N A and APP770 mRNA.

Our p r e l i m i n a r y BamHI-BglII

et al.

analysis

by

Northern blotting

fragment of APP695 cDNA (Fig.

476

I)

has

with

suggested

a shorter that the

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AM-11

>'

AM-13

AM-14

2.5

2.5

2.5

e6

2.0

2.0

2.0

mllm 7

1.5

1.5

r-o~ "o 1.5

*4

"6 *3 e4

~

1.0

o)

e5

lO

el m

0.5

*2 *3

el

el

0.5

e6

e4

0.5

0

I

I

0

CTL

CTL

A 0

I

0

A D

CTL

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 n u m b e r s for w h i c h the densities were calculated (Fig. 2). Bars indicate the m e a n values of relative densities in each group. * p<0.05, Student t-test.

total

amount

disease

of A P P

(unpublished

a significant change the

in A P P 6 9 5

overexpression for

of

of

of A l z h e i m e r ' s by e x o n

J

hypothesis inhibitor J,

with

APP770, of

APP

is n o t

J along

function

of

or w i t h o u t

to

remains

possible

sequence leading

(11,

is

exon

any to

that the I,

of k n o w n

fragment

26),

imposes

to a b e r r a n t

8-protein.

477

of

exon that

I

the

J) e n c o d e d

proteins, An

J affects

and

its

attractive the p r o t e a s e

the f l a n k i n g

by f r a g m e n t

at a t h r e o n i n e a conformational

metabolism

to

to p a t h o g e n e s i s

(fragment

clarified.

that

simply

possible

related

of

by a

suggests

ascribable

peptide

O-glycosylation

finding

expression

is

or t h a t

the

unaccompanied

it s e e m s

be

in A l z h e i m e r ' s

disease,

not

I

acid

to

with

expression

Rather,

with

fragment

together

aberrant

19 a m i n o

is

appreciably

in A l z h e i m e r ' s

homologous

activity

mRNA

inhibitor.

at this m o m e n t

thereby

result,

nor

The

change

8-protein

gene

disease.

the T h r - X - X - P r o

mRNA

amyloid

exon

physiological

This

or APP751 of

not

in A P P 7 7 0

a protease

coexpression

does

data).

increase

deposition

coding

mRNAs

residue

of

change

on

of A P P and d e p o s i t i o n

Vol. 157, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS We t h a n k Drs. M. Ogawa, T. Seko, K. Hara, R. M a t s u m o t o 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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