Expression of the Alzheimer amyloid precursor gene transcripts in the human brain

Neuron, Vol. 1, 669-677,

October, 1988, Copyright 0 1988 by Cell Press

Expression of the Alzheimer Amyloid Precursor Gene Transcripts in the Human Brain be the cause of the amyloid deposits common to Down’s

Rachael 1. New+*+* Elizabeth A. Finch,*+* and Linda R. Dawes*+

syndrome

*Division

however, revealed that the APP gene is distinct from the

of Genetics

The Children’s Boston,

Hospital

Massachusetts

+ Department

02115

and Alzheimer’s

21 responsible

familial

et al.,

Alzheimer’s

disease

(Tanzi

duplicated

Harvard

(Tanzi et al., 1987c; St. George-Hyslop

Boston,

Massachusetts

1987b;

for Van

et al., 1987) and that the APP gene is not

*and Program in Neuroscience Medical School

studies,

the genetic defect on chromosome Broeckhoven

of Pediatrics

disease. Additional

in sporadic

or familial

Alzheimer’s

disease

et al., 1987; Pod-

lisney et al., 1987).

02115

Subsequent

to these findings, a cDNA encoding an al-

ternate form of the APP that contains a protease inhibi-

Summary

tor domain was reported (Tanzi et al., 1988; Kitaguchi et

An alternate form of the Alzheimer amyloid protein precursor mRNA that encodes a protease inhibitor domain has recently been reported. Oligonucleotide probes that differentiate between the two mRNAs are used to describe the expression of each amyloid precursor transcript in the human brain. RNA blot analyses show that one of the mRNAs is expressed selectively in the nervous system, that the two messages display different regional distributions in the adult human brain, and that the expression of the two mRNAs is differentially affected in Down’s syndrome brain and in Alzheimer’s disease frontal cortex. In situ hybridization shows that the two transcripts display the same laminar distribution in the adult cortex but that the transcripts differ significantly in their levels of expression in pyramidal cells of the hippocampus.

tion, we utilize

al., 1988; Ponte et al., 1988). In the present communicaoligonucleotides

of the APP mRNAs

human brain. RNA (Northern) tissue distribution

specific for each form

to characterize

their expression

and overall regional distribution

APP RNAs within the brain; in situ hybridization allow us to identify specific cellular the brain that express the mRNAs. the expression inhibitor tissues.

populations

encoding the protease lacking this do-

reported APP cDNA;

1987) appears to be preferentially is found principally

in neurons of the associative neocor-

the hippocampus,

both APP mRNAs

neuropathology

of Alzheimer’s

disease,

marked by deposits of the proteinaceous

the mRNA

encoding the protease inhibitor

(AD)

is

loid in the walls of the cerebral microvasculature

and in et al.,

neuritic

plaques (Roth

1966; Terry et al., 1981; Whitehouse 198313). Similar

et al., 1982; Glen-

amyloid

the brains of older Down’s syndrome

deposits

occur in

(DS) patients (Mal-

amud, 1972) and, to a much lesser degree, in association

Tissue Distribution of the APP mRNlAs Northern

blot analysis

was used to determine

man fetal tissue distribution (Figure 1). Hybridizations

with cDNA clone FB68L

et al., 1987a) are shown for comparison; bridizes

with both APP transcripts.

oligonucleotide

revealed a 4.2

the amino

kd polypeptide

acid sequence

was obtained

et al., 1988;

that specifically

hybridizes

AMY3 is a 40 base oligonucleotide

Several groups have used this sequence to isolate amy-

insert junctions,

loid protein precursor

transcript

hybridize

with a 3.4-3.6

normally

in the brain and other

that were shown to

kb mRNA

doublet

tissues

expressed

(Tanzi

et al.,

the hybridization al., 1987). FB68L

et al., 1987). These findings suggested the possibility

previously

amyloid deposits in Alzheimer’s an abnormal

modification possibility

expression

of a normal

was strengthened

disease may result from or a posttranslational

molecular

constituent.

This

by the finding that the gene

encoding the amyloid beta protein is found on chromosome 21 (Tanzi

et al., 1987a;

Kang et al., 1987;

Gold-

gaber et al., 1987; Robakis et al., 1987) and hence may

conditions

hybridizes

and HL124i

described

bridize to 3.4-3.6

with the

domain under

used. This AMY3 transcript

to the APP cDNA

1987a; Kang et al., 1987; Goldgaber et al., 1987; Robakis that

with the APP domain (Tanzi

spanning the HL124i

which specifically

lacking the protease inhibitor

corresponds

hy-

is a 22 base

Kitaguchi et al., 1988; Ponte et al., 1988);

(Glenner and Wong, 1984a, 1984b; Masters et al., 1985). (APP) cDNAs

(Tanzi

this cDNA

HL124i

transcript encoding the protease inhibitor

of amyloid

the hu-

of the APP gene transcripts

constituent

either

is

Results

with the normal aging process. Isolation of the principal which

domain

in both normal

material amy-

the core of extracellular

from

are ex-

pressed in the pyramidal neurons of Ammon’s horn, but

adult and aged DS brains.

Introduction

ner, 1983a,

Kang et al.,

expressed in brain and

present at higher levels in these cells

The

within

in the brain and in other

On the other hand, APP mRNA

tex. Within

of the studies

Our results show that

of the APP mRNA

domain is widespread

main (i.e., the initially

in

blots are used to examine

initially

reported (Kang et

tissue expression

has been

(Tanzi et al., 1987a, 1988); both hy-

kb RNA species in all human fetal tis-

sues examined. AMY3 hybridizes

to RNA species of the

same molecular weight (Figure lA), but hybridization

is

only seen in human fetal brain. HL124i

and AMY3 were hybridized

with a second set

of human fetal tissue RNAs (Figure 1B). These hybridizations confirmed

the ubiquitous

nature of the transcript

Neuron 670

HL1241 -

AMY3

-.

FB68L Figure 2. Hybridization Brain

- HL124i

of HL124i and AMY3 to RNA from Adult

HL124i and AMY3 were hybridized to RNA (10 ug) from the brain of a 51-year-old female (PM1 = 2 hr; designation B1154). Cb, cerebellum; Tha, dorsal thalamus; Hi, hippocampus; Amyg., amygdala; AlO, frontal pole of the cortex; A20 and A21, temporal association cortex; A44, anterior perisylvian cortex-opercular gyrus; A7, parietal cortex; A19, extrastriate cortex; A17, striate cortex; Al, primary somatosensory cortex; A4, motor cortex. Exposure time for both hybridizations was 20 hr.

Regional Distribution of APP mRNAs in the Adult Human Brain The same probes were hybridized

to RNAs from differ-

ent regions of the adult human brain (Figure 2). We previously

showed (Tanzi et al., 1987a) that the APP mRNA,

as indicated by hybridization

with FB68L,

shows marked

regional variation in the adult human brain. Expression of the gene is highest in the association cally in Brodmann

contrast, the distribution a-

B

-AMY3

tively homogeneous

HL124i and AMY3 Expression in

transcript

detects both alternate mRNAs

gene, whereas containing

(A) 20- to 22-week fetal tissues; (B) 18-week fetal tissue. All tissue was obtained from midtrimester elective abortuses under protocols approved by the institutional review board at Brigham and Women’s Hospital. Total RNA (25 pg) was loaded in each lane. In (A), the blot to which HL124i and AMY3 were hybridized was equivalenttothat used in the FB68L hybridization. HL124i and AMY3 were successively hybridized to the same RNA blot for (B). The FB68L hybridization was exposed for 16 hr; all other blots shown were exposed for 48 hr.

of the HLl24i

is rela-

across the brain regions (see Tanzi

et al., 1988, and a second case, shown here in Figure 2). Since FB68L

Figure 1. Comparison of FB68L, Human Fetal Tissues

cortex, specifi-

areas AIO, A20/21, A40, and A44. In

HLl24i

mRNA,

hybridizes

the differential

the brain revealed by FB68L due to the APP transcript This

assumption

with the AMY3

only

from the APP

to the HLl24i-

expression

of APP in

was assumed to be primarily lacking the HLl24i

is confirmed

fragment.

by the results

hybridization

shown

obtained

in Figure 2. The

stronger signal in associative areas of the neocortex seen upon hybridization richment

with FB68L

indicates a relative en-

in these regions of the APP transcript

the HLl24i

insert (detected by AMY3).

containing APP mRNA

Notably,

lacking HLl24i-

is expressed at considerably

high-

er levels in the hippocampus than is the mRNA detected by AMY3.

These

patterns

cated in a regional

of hybridzation

analysis

were repli-

of three additional

human brains (see Neve et al., 1988, for FB68L detected by HLl24i

and added meninges,

spinal cord,

zations;

data for AMY3

and HLl24i

adult

hybridi-

are not shown).

It

and cerebellum to the tissues found to express this RNA.

should be noted that although the case shown in Figure

The AMY3 transcript

was additionally

2 displays

cord and cerebellum

but was not found in any other tis-

sue, including bridization

meninges.

detected in spinal

Note the lack of positive hy-

to kidney RNA in Figure IB; the band seen

a relative abundance of the HLl24i

hippocampus

contrast was not evident in other individuals. To quantify our observations, analysis

nonspecific

hybridization

to an overloaded

lane. Thus,

the RNA to

ond protease

which

hybridizes,

that is, the RNA

lacking the

tide, HLl25i,

protease selectively

inhibitor

domain,

in the nervous

appears to be expressed

system.

inhibitor which

transcript.

domain-specific

oligonucleo-

was 40 bases in length.

blot analysis confirmed HLl24i

we carried out a slot blot

in which we used as probes AMY3 and a sec-

in kidney RNA in Figure IA may represent AMY3

RNA in

compared with other brain regions, this

the specificity

of HLl25i

Both oligonucleotides

Northern for the

were radiola-

Alzheimer Amyloid Precursor Gene Expression 671

beled to a specific activity of 5 x 1Oacpmlwg and were hybridized to a slot blot containing RNAs from hippocampus, frontal cortex (AlO), and inferior temporal cortex (A20) from four different adult brains. Densitometric analysis revealed that the relative abundance of the AMY3 transcript in these regions was 1:2.5:2, respectively; for the HL125i transcript, it was lZ1.2:l.l. Acrosscomparison of the two RNAs showed that the abundance of AMY3 RNA relative to HLlESi RNA in each region was 0.6, 1.4, and 1.2, respectrively. These data confirm the qualitative impression derived from the Northern blot analyses.

-IiLl

Figure 3. Hybridization DS and AD Brain

of HL124i, AMY3, and FB68L to RNA from

(A) Northern blots of HL124i and AMY3 hybridizations to total mRNA (25 pg) from 19-week normal (N fetal) and trisomy 21 (DS fetal) brains, adult normal (N cb) and AD (AD cb) cerebellum, and adult normal (N ctx) and AD (AD ctx) frontal cortex. Fetal tissue was obtained from an abortus with a diagnosis of Down’s syndrome and from an age-matched normal abortus. Adult tissue was obtained from autopsy brains of a case of histologically confirmed Alzheimer’s disease and from an individual without dementing illness. Control hybridization with a cDNA for the microtubule-associated protein tau (Neve et al., 1986) is shown above. Tau gives a pattern of hybridization in normal versus AD cortex that is typical of a number of cDNAs we have tested, both neuron-specific and otherwise, and that probably reflects the extensive neuronal loss in AD frontal cortex. The three autoradiograms are the results of independent hybridizations with the same filter. RNA isolation and hybridizations were performed as described in Figure 1. Exposure time for the AMY3 blot was 48 hr.; for the HL124i blot it was 72 hr. (B) Hybridizations of FB68L, HL124i. and AMY3 to RNA (10 pg) from the brain of a 37-year-old DS female (PM1 = 23.6 hr; 81037). Cb, cerebellum; C-e caudate putamen; Hi, hippocampus; AlO, frontal pole of the cortex; A20, temporal association cortex; A40, posterior perrsylvian cortex-supramarginal gyrus; A44, anterior perisylvian cortex-opercular gyrus; A6, supplementary motor cortex; A7, parietal cortex; A19 and A18, extrastriate cortex; A17, striate cortex; Al, primary somatosensory cortex; A4, motor cortex. The brain was confirmed upon autopsy to display the neuropathological characteristics of aged DS brains (neuritic plaques, neurofibrillary tangles, and cerebrovascular amyloid). A second DS brain displaying similar patterns of hybridization, was from a 57-year-old DS female (PM1 = 13.2 hr; 8937). The FB68L blot was exposed for 20 hr. the HLl24i blot for 5 days, and the AMY3 blot for 7 days.

APP Expression in Down’s Syndrome and Alzheimer’s Disease HL124i and AMY3 were hybridized with RNAs from brain tissue of individuals with Down’s syndrome and Alzheimer’s disease (Figure 3A); hybridizations were compared with those using a cDNA fot the microtubuleassociated protein tau (Neve et al., 1986). It is clear that in 19-week fetal brain the AMY3 transcript is expressed normally at higher levels than the HL124i transcript. The intensity of HL124i and AMY3 ‘hybridizations to mRNA from a 19-week DS fetal brain are both increased several-fold relative to hybridizations with RNA from the normal 19-week fetal brain, indicating that both APP transcripts are overexpressed in DS brain. Hybridizations of HL124i and AMY3 to adult normal and AD cerebellum, a region relatively spared in Altheimet’sdisease, show that both transcripts are present, at approximately normal levels in AD cerebellum. Hotiever, HL124i and AMY3 hybridizations to normal and AD frontal cortex reveal striking differences in the expression of the two transcripts. The level of HL124i mRNA is near normal, whereas the AMY3 transcript appears to be selectively lost in AD frontal cortex. This loss may be due to death of the neurons that normally express the AMY3 transcript, or to decreased expression of this transcript in affected regions of the AD brain. Thle level of HL124i expression could appear relatively unaffected in AD if the cells expressing this transcript are spared or if the transcript is overexpressed to the extent that, despite loss of cells expressing HL124i RNA, a significant level of the message remains. The patterns of expression of AMY3 and HL124i transcripts shown here were confirmed in two additional cases of fetal DS brailn relative to agematched controls and in two additional cases of AD cerebellum and cortex compared with adult controls. We again quantified our results by hybridizing all three blots (i.e., the blot shown in Figure 3A and the two blots with additional cases that are not shoivn) to AMY3 and then to Hl125i (both labeled to a specific activity of 5 x 1Oacpmlpg) and performing dengitometric analysis on the hybridization signals that we obtained. The average abundance of AMY3 RNA in DS fetal brain compared with normal was 4.6; that of HL125i RNA in DS relative to normal was 3.8. For both, transcripts, there was no significant difference in levels between normal and AD cerebellum. The average ratio for AMY3 RNA in normal relative to AD cortex was 3.5; for HL125i RNA it was 1.5.

Neuron 672

A4

I

‘. ‘.

Figure 4. Expression

of APP mRNA

in Adult

Human

Cortical

Subregions

as Determined

*. . I. fl

,

by In Situ Hybridization

In situ hybridization was performed with an antisense RNA probe transcribed with SP6 polymerase from a 700 bp EcoRI-Hindlll subfragment of FB68L in pCEM3. A17, striate cortex (primary visual area); A20, inferior temporal cortex (association area); A10, frontal cortex; A4, motor cortex-precentral gyrus (primary motor area); A40, posterior perisylvian cortex-superamarginal gyrus. Roman numerals indicate the cortical

Attempts to do a regional analysis of APP expession AD brains were unsuccessful

in most cortical areas. However,

surveys of two aged DS

brains

(ages 37 and 57 years) were carried

former

is shown

in Figure 3B. The expression

RNA appears relatively

in

due to degradation of RNA out; the of HL124i

unaffected in aged DS brain; it

shown in Figure 2. Both the strength and the laminar pattern of hybridization

in agreement with Northern

cortical

blot data. A4 and A40 dis-

play moderate levels, and Al7 hybridization.

relatively

The laminar distribution

is seen at high and relatively invariant levels across brain

homogeneously

regions. The AMY3 RNA is normally

II-VI

present at relatively

vary among the different

areas. A20 and A10 exhibit highest APP gene expression,

strong hybridization

low levels, of varies from the

throughout

layers

seen in the associative areas A20 and AlO, to the

high levels in associative neocortex (AlO, A20, A40, A44,

more striking

A6, and A7); however, in both aged DS brains, its expres-

plays strongest signal in layers III and V. A17, primary vi-

sion is depressed in these areas compared with normal.

sual

cortex,

variable laminar pattern in A4, which diswhich

contains

densely

packed granule

cells and relatively few pyramidal cells, exhibits weak hy-

localization of APP mRNAs by in Situ Hybridization To analyze the distribution man brain regions

bridization

in the Adult Brain

predominantly

of APP mRNAs

by in situ hybridization,

in adult huwe initially

To differentiate

(Tanzi et al., 1987a). Note that this hybridization

oligonucleotide

confirm

reveals

The results (Figure 4)

and expand the RNA blot analysis

with FB68L

neurons

express

between the patterns of expression

the two APP transcripts

of both APP transcripts.

pyramidal

the highest levels of APP mRNA.

used an RNA probe transcribed from a subclone of FB68L expression

in layers III and V/VI. These

data suggest that in cortex,

probes HL124i

in situ hybridizations. temporal

described

of

above, we used the

and AMY3 in subsequent

Hybridization

cortex of 1% to 20.week

of the probes to the fetal brain is shown

Alzheimer 673

Amyloid

Precursor Gene Expression

Figure 5. In Situ Hybridization

of HL124i and AMY3

(A) Bright-field, HL124i; (B) dark-field, compared with HL124i

in Figure 5. The higher levels of AMY3 HL124i

to 19- to 20-Week Temporal

HL124i; (C) bright-field,

AMY3;

APP RNA than

RNA in fetal brain revealed first by RNA blot anal-

ysis (Figure 3) are dramatically

confirmed

by in situ hy-

In contrast, the two transcripts lar pattern of exression

appear to have a simi-

in the adult cortex. Northern

revealed that the AMY3

higher levels than the HL124i

mRNA

mRNA

blot

is expressed

at

in associative neo-

Cortex AMY3.

date-putamen, bution

of cells expressing

mRNA

is evident in both aged DS brain

(Figures 6A and 66) and control adult brain (Figures 6C

reflected in the observed heavier labeling of many corti-

and 6D). The contrast

of the observation that HL124i

relative to HL124i.

The laminar distribution and mirrored antisense

that shown

RNA,

is particularly

of each transcript was similar,

pyramidal cells in the cortex is considerably than AMY3

which detects both mRNAs

(Figure 4).

We have observed bridize

cells in layer V compared with layer II for HL124i

their expression

in A44

problematic.

In A4, the V:ll

ratio for HL124i

mRNA

was 1.5. While

the hybridization

was 1.8; that for AMY3 for each probe is not

tissue,

that both HL124i

to a variety of neuronal

was 2.0; the same ratio was observed for AMY3 in A44.

in nonneuronal

RNA blot analysis

is robustly

expressed

while AMY3

hybridization

clearly that neither APP transcript

neu-

ing to their cytoarchitecture. strikingly

The differential

levels of

of the two APP messages are revealed most in subcortical

areas by both RNA blot analysis

hyof

reveal’s that the HL124i

in human fetal meningeal

ing reflects the laminar

of pyramidal

and AMY3

cells in the brain is more

not detectable. Our

distribution

less intense

types. The question

restricted to pyramidal cells, the overall pattern of labelrons in the cortex, which varies among regions accord-

in light

to individual

hybridization.

For example, the ratio of density of grains over pyramidal

expression

significant

hybridization

by the hybridization

with the

tran-

in pyramidal

mRNA in these hippocampal neurons rel-

cal neurons

by AMY3

with the HL124i

specifically

cells of Ammon’s horn (Figure 6). The increased expression of HL124i

but it may be

of both

these messages in the hip-

being more abundant

ative to AMY3

readily apparent with in situ hybridization,

of HL124i

and diffuse. The distri-

pocampus is more circumscribed, script

cells

in the adult cau-

where the pattern of expression

seems to be widespread

pattern across cortical regions. This

was not

In situ hybridization

is much higher than that of AMY3

cortex; the latter message displays a less heterogeneous difference

Note the increased density of AMY3-labeled

and in situ hybridization.

mRNAs

bridization.

anaysis

(0) dark-field,

to meningeal

in situ hybridization

RNA is

data indicate

is expressed in the en-

dothelial cells lining blood vessels in the brain (Figure 7). Neither the antisense RNA probe that detects both messages (Figure 7A) nor either of the oligonucleotide (AMY3 shown in Figure 7B) hybridizes

probes

to these cells. Fig-

HL124i

DS

Figure 6. In Situ Hybridization

of HL124i and AMY3

to Pyramidal

Cells in DS and Control

Hippocampus

(A and B) Hippocampus from a 37-year-old patient with Down’s syndrome. (C and D) Hippocampus individual. HL124i hybridizes more intensely to these neurons than does AMY3.

ure

7C illustrates

cell

types in A44;

of AMY3

hybridization

HL124i

similarly

cells besides pyramidal

neurons.

cell types have been definitively ronal. Although

to a variety

is expressed

None of these other identified

as nonneu-

we cannot rule out the possibility

one or both of the APP transcripts

is expressed

neuronal

neurons

cells

in the brain, only

been positively

identified

in the cerebellum, affected in Alzheimer’s

of

in other

as expressing

that

in non-

have so far

a region of the brain largely undisease, the most striking

ization of both probes is to Purkinje

surveys of a number of tissues,

hybrid-

cells in the molecu-

neurologically

normal

using RNAase protection

assays, will be required to determine whether this mRNA is expressed exclusively below the sensitivity AMY3 transcript deposition

in the brain or is present at levels

of RNA blots in other tissues.

If the

is indeed made only in the brain, the

of amyloid deposits exclusively

in Alzheimer’s dividuals

APP RNAs.

from a 56-year-old

in this organ

disease and in aged Down’s syndrome

may be the result of an abnormality

only the protein made by this mRNA.

in-

affecting

The increased ex-

pression of the AMY3 mRNA in associative neocortex relative to primary

sensory

cortex is exemplified

lar layer (Figure 7D).

atively higher hybridization

Discussion

cies may contribute

by its rel-

signal in A20 compared with

Al7 (Figure 2; see also Figure 4, in which this RNA speto the inceased density of grains in

A20 compared with A17). This regional variation parallels Previous

studies that described the in situ hybridization

the regional distribution

of neurofibrillary

tangles found

pattern delineating AAP gene expression

in the human

in visual cortices of AD brains (Lewis et al., 1987). Al-

brain (Bahmanyar

1987) utilized

though this same study showed that the number of neu-

molecular

et al., 1987; Coedert,

have devised oligonucleotide tween the two mRNAs. cDNAs

We

ritic plaques did not vary between primary and associa-

be-

tive visual cortical regions, earlier studies have postulated

to the

a greater number of neuritic plaques and cerebrovascu-

probes common to both APP transcripts.

originally

probes that distinguish

The mRNA corresponding

isolated (Tanzi et al., 1987a; Kang et

lar amyloid deposits in association than in primary sen-

al., 1987; Goidgaber et al., 1987; Robakis et al., 1987),

sory

i.e., the mRNA

Vinters

lacking the protease inhibitor

shown to be expressed

preferentially

domain, is

in brain. Rigorous

areas (Mlandybar, and Gibert,

1975;

Morimatsu

et al.,

1975;

1983).

The laminar distribution

of the two APP mRNAs

within

Alzheimer Amyloid Precursor Gene Expression 675

Figure 7. In Situ Hybridization

of APP Antisense RNA and AMY3 to Neuronal and Nonneuronal

Ceils

Hybridization of APP Antisense RNA (see Figure 4) and AMY3 to areas around blood vessels in the adult brain (A and 6). No hybridi zation is seen to endothelial cells lining the blood vessels. (C) Hybridization of AMY3 to pyramidal and other cell types in A44. (D) Hybridi zation of APP antisense RNA to Purkinje cell in the cerebellum.

the cortex is very similar.

It varies among regions in a

manner parallel to the distribution al cells in the cortex. mRNA

expression

Although

the lamination

is not well defined,

clearly expressed preferentially and the distribution of neocortical studies

of the large pyramid-

plaques

(Pearson et al., 1985;

Both RNA blot analysis

roughly

AMY3

Duyckaerts

mRNA

show

also displays

significant

Dyrks

changes during

cell-cell

et al., 1988).

et al., 1986).

is expressed

mRNA

drop in

levels do not

development.

is a cell surface

interactions

(Shivers

Such a protein would

remore

message. The

a developmental

the HL124i

been suggested that the APP mediating

in several

and in situ hybridization

in fetal brain than the HL124i

abundance, whereas

parallels that

as reported

sults reveal that the AMY3 transcript vigorously

it is nevertheless

in certain cortical layers;

of the mRNA

senile

of APP

et al.,

It has protein 1988;

be expected

to be present in abundance in the developing compared with the adult brain and may be encoded specifically the AMY3 transcript. process outgrowth

depend on a balance between the

two APP gene products, tion to maturity.

by

Perhaps rates of cell migration and which shifts during the transi-

Overexpression

of both APP messages

in DS fetal brain, which we demonstrate rupt these developmental

here, could dis-

processes.

It is intriguing that the AMY3 mRNA seems to be preferentially

lost in AD frontal

cortex and in association

areas of aged DS brain (Figure 3), whereas the HL124i mRNA

is comparatively

The decrease in AMY3

unaffected in these same areas. hybridization

suggests that cells which are lost,

on the RNA blots express

perhaps due to some stimulIus

production is difficult

normally

or down-regulation to determine

by RNA

this mRNA

causing over-

of the AMY3 blot analysis

mRNA.

It

whether

NWKM-

676

AMY3

mRNA

expression

is depressed in AD or DS hip-

pocampus, since it is normally than HL124i

mRNA

in this region of the brain. Careful

in situ hybridization expression

expressed at lower levels

analysis

of the level and pattern of

of these two transcripts

in the associative cor-

tex and hippocampus of normal and affected brains may shed some light on the participation

of the APP gene

products in the process of neuronal degeneration in Alzheimer’s disease and Down’s syndrome. Experimental

Procedures

Oligonucleotide Probes The 22 base oligonucleotide termed HL124i is homologous to a relatively nonconserved portion of the nucleotide sequence encoding the protease inhibitor domain present in the previously described alternate APP message (Tanzi et al., 1988). The sequence of HL124i is 5’-CATCCACTACTCTTCTCTGTCA-3’. The 40 base oligonucleotide termed AMY3 encompasses 20 bases on either side of the potential splice junction sites in the cDNA HL124 (Tanzi et al., 1988), which includes the protease inhibitor domain; it encompasses 40 contiguous bases in cDNAs lacking this domain. The sequence of AMY3 is 5’-CTCGCTGCTGTTGTAGGAACTCGAACCACCTTTCCACAGA-3’. For quantitative analysis, a 40 base oligonucleotide specific for another nonconserved region of the protease inhibitor domain, termed HL124i, was synthesized. The sequence of HL125i is S’-HCTTCCCTTCAGTCACATCAAAGTACCAGCGGGAGATCAT-3’. RNA Blot Hybridization Oligonucleotides were 32P-labeled using T4 polynucleotide kinase (BRL). The specific activity of both radiolabeled oligonucleotides was consistently 2 x 10s to 4 x lo* cpmlpg. Hybridizations with HL124i were carried out in 5x SSC (lx SSC = 0.15 M sodium chloride, 0.015 M sodium citrate), 50% formamide at 30°C, followed by three 30 min washes in 3x, 2x, and lx SSC at room temperature. Hybridizations with AMY3 were carried out in the same buffer at 37°C and were washed two times for 20 min each at 58’C in 3 M tetramethylammonium chloride, 2 mM EDTA, 50 mM Tris (pH 8.0). Methods of RNA isolation and hybridization with FB68L have previously been described (Tanzi et al., 1987a). Blots were exposed to Kodak X-Omat AR film. For slot blot hybridization, RNA samples (2 pg) were vacuumdried, dissolved in 50 ul of 6.1 M formaldehyde in 10x SSC at 65°C for 15 min, and brought to a volume of 200 ~1 with 15x SSC. Biotrans membrane was prewetted with 10x SSC and placed on a slot minifold apparatus (Schleicher and Schuell). Samples were loaded and vacuum-applied. Filters were baked under vacuum at 80°C for 1 hr, and the RNA was cross-linked to the membrane by exposure to UV light for 2 min. Hybridizations were performed as described for AMY3 Northern blots, except that the hybridization temperature was increased to 42°C. Radioactive signals from blots were estimated with the LKB Ultroscan XL soft laser scanning densitometer. Areas under optical density peaks over a path encompassing the length of the entire slot or lane were measured. Exposure time for all slot blots was 60 hr. Exposure time for the Northern blots on which densitometric analysis was performed (see Results) was 48 hr. In Situ Hybridization Human brains from two adults with no neurological disorders (56 year-old-male, postmortem interval [PMI] = 12.5 hr; designation 81047; 51-year-old female, PMI = 2 hr; 81154) and from a 37-yearold patient with Down’s syndrome (81037; described in Figure 3) were analyzed. Brain tissues were immersion-fixed in 4% paraformaldehyde (PFA) for 24 hr, cryoprotected in buffered 30% sucrose (4°C) for 2 days, and stored at -70°C until being sectioned. Brains were cut at -22°C at lo-15 pm intervals. Sections were mounted on microscope slides coated with TESPA (3-aminopropyltriethoxysilane, Pierce) and activated with 4% PFA and stored dessicated at -80°C. Radiolabeled ([?S]UTP Amersham, 800-1000 Ciimmol) RNA

was synthesized from the FB68L subclone in pGEM-3, using Promega Biotec protocol, to a specific activity of lo9 cpm per pg of template. Tissue sections were rehydrated through graded ethanols, pretreated in 20 mM HCI, 0.01% Triton X-100, 1 ug/ml proteinase K, postfixed with 4% PFA, and acetylated by immersing the slides in 100 mM triethanolamine (pH 8), 0.25% acetic anhydride and stiring for 10 min. After the sections were rinsed in PBS with 2 mg/ml glycine, they were prehybridized in 50% deionized formamide, 2x SSC, 25 &ml yeast tRNA, 250 us/ml salmon testes DNA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin, 0.2% SDS, 25 mM EDTA for 1 to several hours at 55°C. The prehybridization mix was drained from the slides, which were then incubated with hybridization buffer (prehybridization mix plus 1 x lob cpm of probe per 75 ul] at 55OC overnight. After hybridization, sections were washed in 2x, lx, and 0.1x SSC for 30 min each at room temperature, then in 0.1 x SSC at 42OC, 55”C, and 65’C. Finally, they were treated with l-20 &ml RNAase A in 2x SSC at 37OC for 30 min, washed in 2x SSC at 6S°C for 15 min, air-dried briefly, dehydrated through graded concentrations of ethanol, and dipped twice in xylene. The sections were air-dried, dipped in Kodak NTB2 emulsion, and exposed at 4OC for 3 days to 3 weeks. Slides were developed in Kodak D19 developer and fixed in Kodak fix; the sections were counterstained lightly with 0.1% cresyl violet. Hybridization was observed using both bright-field and dark-field microscopy, and Kodak Panatonic-X (continuous tone) film was used for photography. Oligonucleotide probes were labeled by tailing with [‘%]dCTP (Amersham, >lOOO Cilmmol) using terminal deoxynucleotidyltransferase according to the protocol recommended by the supplier (Bethesda Research Labs). Hybridization with the oligonucleotides was carried out in hybridization buffer identical to that described above. except that the salmon sperm DNA was removed, the salt was raised to 5x SSC, and 10% dextran sulfate was included. HL124i was hybridized at 30°C and washed at 37°C in 2x SSC (four 15 min washes) and then lx SSC (four 15 min washes); AMY3 was hybridized at 37°C and washed at 5S°C in 2x SSC (four 15 min washes) and then lx SSC (four 15 min washes). Under these conditions, as verified by RNA blot analysis, the oligonucleotide probes hybridized only to APP mRNA species and displayed no nonspecific hybridization to ribosomal RNA. Pretreatment of sections with RNAase A abolished specific hybridization of probes to cells. All oligonucleotide in situ hybridization was photographed with Kodak Tri-X pan film and developed with Kodak HCllO (dilution B).

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