A distinct form of tau is selectively incorporated into Alzheimer's paired helical filaments

Vol. 159, No. 3, 1989 March 31, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

1221-1226

A DISTINCT FORMOF TAU IS SELECTIVELYINCORPORATED INTO ALZHEIMER'S PAIREDHELICAL FILAMENTS Hiroshi Mori,

Yoshio Hamada*, Masahiro Kawaguchi*, Toshiyuki Honda**, Jun Kondo**, and Yasuo Ihara +

2nd Laboratory of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173, JAPAN *

National Institute **

for Basic Biology,

Okazaki, Aichi 444, JAPAN

Mitsubishi Kasei Corporation Research Center, Biosciences Laboratory, Kamoshida, Midori-ku, Yokohama 227, JAPAN

Received February 8, 1989 SUMMARY: Tau, a microtubule-associated phosphoprotein, was identified as a definite component of paired helical filaments which progressively accumulate in Alzheimer's disease brain. To learn more about tau in the aged brain, we have isolated and sequenced a cDNA clone encoding tau from a cDNA library of The cloned cDNA sequence included a new insert of 93 an aged human brain. nucleotides, which added a fourth repeat to the three-repeat type of tau Perhaps, this four-repeat type of tau is predominant in already reported. In contrast, the sequence analysis of paired helical normal aged brain. filaments showed that the integrated tau is of three-repeat type. This indicates that a distinct form of tau is selectively incorporated into paired 0 1989Academic Press.Inc. helical filaments.

The progressive paired helical (AD).

accumulation

filaments

Neurofibrillary

restricted

II

of

cortex,

These tangle

progression of AD (4,5). turn, mitters, +

pyramidal

and IV of the entorhinal in

particular

cells

cortex,

in CA1 of

and layers

The loss of these particular alterations

which are considered to be closely

III

temporal and parietal

bearing neurons are indeed the ones lost

causes the significant

disease

bundles of PHF, appear to be formed in a

of neurons including

hippocumpus, layers

(1,2,3).

designated as

(PHF), is one of the hallmarks of Alzheimer's tangles,

population

the association

of unusual neuronal fibers,

of

the linked

the

and V

cortices during

the

subsets of neurons, in

levels

of

several

to the clinical

transmanifes-

To whomcorrespondence should be addressed. ooc6291x/89 $1.50 1221

Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 159, No. 3, 1989

tations

of

AD.

understand

PHF on the

of

(11,12).

antibodies

nature

that

are

of

going

reactivities

addition,

immunochemical to

tangles

in

tau

to

neurons,

as a component

(6-10)

and

studies

(14)

is

the AD brain.

was identified

immunological

or a monoclonal

of

tangle-bearing

to die

phosphoprotein,

In

to PHF (13)

the

on

microenvironments

of neurons,

basis

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

investigation

microtubule-associated

sequencing

later,

protein

using

showed

polyclonal

that

the

of

its

tau

in

PHF

phosphorylated. Tau

undergoes

forms:

tau

bands

with

is

remarkable

present

and their

appears

In fact,

carboxyl

terminals.

show

will here

instead

the

arise: presence

of three-repeats

three-repeat

distinct

form

one

band

in

al.

fetal

stage

from

In

view

Is

any in

based is

showed

of

such

specific aged

two

and

form

human

brain

integrated

our

MATERIALS

becomes

of

isoforms

several

tau,

of

tau of

of

tau,

incorporated tau

data

(12),

(15).

isoforms phosphory-

short

and long

an

important

into

PHF?

containing the

several

stage

in particular

Furthermore,

previous

into

of

molecular

the adult

presence

heterogeneity

reported. on

the

modifications,

(16)

already

type, of tau

alterations

the

to originate

posttranslational Lee et

lation.

developmental

mass of 50 - 70 kD on SDS-PAGE in

subsequent

question

as

a molecular

Such heterogeneity

of

the

intracellular

subsets

Tau,

is

Thus

the

particular

of

BIOCHEMICAL

four-repeats

tau within indicating

We

PHF is that

a

PHF.

AND METHODS

Preparation

of adult human brain cDMA library: mRNA was prepared from frontal cortex from a 82-year-old patient without dementia by a phenol extraction method and poly(A)+RNA was purified by oligo (dT) column Double stranded cDNA was synthesized (18) for the adult chromatography (17). human poly(A) RNA, methylated with EcoRl methylase and linker-ligated into the EcoRl site of the expression+vector, lambda gtll or lam da gtl0. We obtained from about 0.1 ug of poly(A) RNA a library plaques, of 1.2 x 10 B independent 80% of which carried cDNA inserts overlapping about 1.0 kbp. cDNA cloning: The recombinant clones expressing B-galactosidase-tau fusion proteins were detected with anti tau C4 antibodies that were raised against IGSLDNTTHVPGGGNK (tau C4). a peptide sequenced from the PHF digest (12).

pieces of

We initially screened about 6 x lo5 plaques of lambda gtll from the amplified library and identified immunologically 12 positive signals. One of these clones named S8 carrying 0.3 kbp insert was used as a starting clone. We screened about 2 x 106 plaques of lambda gtl0 by plaque hybridization with S8 labeled by random-primer technique (19) and isolated 10 clones. Two (T-9 and T-10) of these were subcloned into pUC18 or pUC19 plasmids for further analysis.

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BIOCHEMICAL

Vol. 159, No. 3, 1989

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

DNA sequencing: The EcoRl inserts were digested with Find III and subcloned into M13mp18 or Nl3mp19 (20). The nucleotide sequence of the single stranded phage DNA was deteriined by the dideoxy chain termination methoh (21) with Neuclcotide and amino acid sequences were analyzed with Sequenase (USBC). programs f,rom the GENETYX SOFTWARE PACKAGE (SOF'IWARE Development Co., Japan).

RESULTS AND DISCUSSION We constructed clones

encoding

human brain (ii)

adult

and do not

PHF formation

isolated

and

includes

n new

amino

acid

sequences

occur

in other

to

(Fig.

2).

point

is

end

of

carboxyl

end

of

Achmobacter

and

III

(Fig.

PHF digest

(12)

the II

site

3). is

I

absence

of

and I

between

The peptide the

tau

amino

before II

are

(APl),

the

already

to

carboxyl

composed

for

primates

and

aging.

We

tau,

which

sequence

of

the

o: tandem

arrangement

is

lysine

peptide

III

(Fig.

lysine

and

serine,

of VOIVYKPVDLSI: of

highly III

but

that peptide

of

I +

residue

at

the

3j.

Since

the

respectively, for

between

was sequenced III.

of

composed

specific not

(16)

Thus

Lys-X

peptides from

the

the

tau

TETGECT;AAflGG;TA;TA;CAAACACGTCCCGGGAGGCGGCAGT KHVPGGGS

Figure 1. Sequence analysis of a new insert. acid (lower) numbering Is based on the sequence 1223

31

three

of mouse

reported

I and

other

half

a particular

portion

specific

of

The

obtain

~'qIINKKLDLSIIVOSKCGSKD~IIKHVP-

a protea?e

peptides

terminal

1).

to

normal

form

homologous

in is

the

just

protease the

IV)

is is

in

another (Fig.

insert

highly nonhuman

phenomenon

which

III,

while

peptide

lyticus

can #cleave

(23),

11) (I,

peptide

including

encoding

this

human brain

PHF are

93 nucleotides

from

t- IV,

normal

(i)

mammals

The tau we sequenced

+ IV (16,22)

carboxyl

of

repeats

+ III

crucial

sjnce

clone

peptide

1 + II

The

an aged

age-dependent

a cDhA

as

(22) I *

tau,

a highly

deduced

contafning

peptides

of

cDNA inrcrt

residues

and human tau

III

is

from

forms

sequenced

GGGS (referred

II

a cDNA library

Nucleotide (upper) and amino of human fetal tau (22).

Vol.

159,

No.

3, 1989

BIOCHEMICAL

AND

1%

I

BIOPHYSICAL

COMMUNICATIONS

216

QTAPVPMPDLKNVKSKIGSTENLKHQPGGGK Al

II

A31

A917

VQIINKKLDLSNVQSKCGSKDNIKHVPGGGS

four repeat

217 VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ

III

iv

‘&KSEKLDFKDRVQSK

type ...‘.....

I GSLDN

228

PGGGSVQ

247

I VYKPVDLSK

216217

three

02

RESEARCH

repeat

03

I THVPGGG?

228

type ~~~~~~.~. PGGG API

Four tandem-repeated sequences in a new type of tau. Three repeats Figure 2. (16) correspond to amino acid residues, 198in mouse tau shown by Lee et al. 215, 229-246, and 261-278, while three repeats in human fetal tau proposed by Goedert et al. (22) are made of 31 or 32 amino acid residues, 198-228, 229260, and 261-292. Here a new insert (peptide II; Al - A31) is regarded as one Thus a new type of tau contain peptides I + II + III + IV unit for repeat. instead of peptides I + III + IV reported in human fetal tau (22). Figure 3. The presence III removes the cleavage amino acid of peptide II repeat type of tau. The

within

PHI'

question

is

of

is:

and

constructed

from

respectively. species

brain.

Kosik

and

types

repeat

brain:

this

and adult on

(24), in

of

fetal

from form

of

never

aged

the

aged tau

in

observed

aged in

could

we

aged tau

been

using

brain,

a predominant in

adult

of

tau

is

type

c;f

tau

is

or

substantiated

by

juction-specific

type

three-

is

tau?

libraries

human

dominates

recently

show

can

tau,

oligo-

expressed

only

specific

and four--repeat

assume

is

fetal

for types

of

sequence

would nammals.

1224

expression

tau

in

adult of

to

thj

appears

tau

neurofibrillary

three-

PHF formation. from

rat

to be expressed the

digest

s unusual

PHF formation

Numerous

of

different

VQIVYK from

Perhaps, lead

for

to be rather

type

(12).

that

a prerequisite

appears

we could

other

tau

ONA

of

a

respectively.

of

brain

of

of

type

from an

type type

us to call

brains

and

Now

type.

four-repeat

cloned

(22)

has very

situation

since

control

were

four-repeat

human brain

amount

tau

three-repeat

type

four-repeat or

three-repeat

of tau,

in human brain

prepared

rat

of

three-

brain

observations,

tau,

of

who

while

types

not

of

speculation

allows

these

but

four-repeat

while

brain,

in a significant

is

that

that

in normal

fetal

suggests

observation

type

However,

human

colleagues

This

fetal

fetal

brain,

prohes

Based

that

four-repeat

and neonatal

brain. as

significance

Indeed, his

type

the

a

fetal

nucleotide fetal

is

This

in

aged

three-repeat

what

Three-repeat

of a new insert (peptide II) between peptides I and site by APl (arrowhead), since the carboxyl terminal is serine (upper) instead of lysine (lower) in threepeptide sequenced from the PHF digest is boxed.

expression in

human tangles

of

tau of

brain in

Vol. 159, No. 3, 1989

the

AD brain

unusual

indicate

that

resistance

presence

of

antigen

(25)

speculate

in reflect

of

(Y.I.!. in

other

and

the

a fetal

an active

should us

to

two

that

possibly

antigen

BIOCHEMICAL

abnormal

type

proteases.

of

antigens

fetal

gangljoside,

form phase neurite

of

tau

is

However,

fetal

Presumably, the

fetal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tau

of AD.

abundant,

when

we

associated

is The

at

reexpression

three-repeat

the

type

tau

intc

it

account

is

the

its the

Alz

reasonable

high

level

as well,

of

these

fetal

antigens

(3)

observed

is

one

of

such

50 to

the

AD brain of

due to

tangles;

(261,

expressed

in

take

with

CQlc

outgrowth

possibly

by one fetal

AD brain.

ACKNOWLEDGMENTS We thank Dr. K.S. Kosik for his helpful comments on our manuscript. This work was supported in part by grants from the Ministry of Education, Science and Culture, the Mjnistry of Health and Welfare, and the Uehara Memorial Foundation., REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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Watson, C.J., and Jackson, J.F. (1985) DNA Cloning --- A Practical Approach IRL Press, Oxford. Feinberg, A.P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. Messing, J. (1983) Meth. Enzymol. 101, 20-78. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. Goedert, M., Wischik, C., Crowther, R.A., Walker, J.E., and Klug, A. (1988) Proc. Natl. Acad. Sci. USA 85, 4051-4055. Masaki, T., Fujihashi, T., Nakamura, K., and Soejima, M., (1981) Biochim. Biophys. Acta 660, 51-55. Kosik, K.S., Orecchio, L.D., and Neve, R.L. (1989) Neuron in press. Wolozin, B., Scicutella, A., and Davies, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6202-6206. Emory, C.R., Ala T.A., and Frey II, W.H. (1987) Neurology 37, 768-772.

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