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Cerebral cortical amyloid protein precursor mRNA expression is similar in Alzheimer's disease and other neurodegenerative diseases
Journal of the Neurological Sciences, 111 (1992)33-38
33
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03800
Cerebral cortical amyloid protein precursor mRNA expression is similar in Alzheimer's disease and other neurodegenerative diseases Yasumasa Ohyagi
a,1, Keikichi T a k a h a s h i
a, Y u h j i S a t o h b, T a k a o M a k i f u c h i c and Takeshi Tabira a
a Division of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan, b Hizen National Mental Hospital, Kanzaki, Saga, Japan, and c National Saigata Hospital, Ogata-town, Niigata, Japan
(Received 6 December, 1991) (Revised, received 16 February, 1992) (Accepted 15 February, 1992) Key words: Amyloid protein precursor mRNA; Alzheimer's disease; Neurodegenerative disease; Gliosis; Neuronal loss
Summary The expression of 3/3-amyloid protein precursor (APP) mRNAs (695, 751, and 770) in the cerebral cortex in Alzheimer's disease and other neurodegenerative diseases was analyzed by the SI nuclease protection assay. We found no significant Alzheimer's disease-specific alteration of APP mRNA expression when compared to the other neurological diseases as controls. Since the expression of this mRNA was not correlated with amyloid deposition, it is possible that gliosis/neuronal loss may secondarily alter APP mRNA expression. However, the current study revealed no significant correlation between them.
Introduction
In the Alzheimer's disease (AD) brain, large numbers of senile plaques are found throughout the neocortex and hippocampus. Beta (or A4)-protein is the major component of senile plaque amyloid and is thought to be derived from amyloid protein precursors (APP) (Kang et al. 1987). At least 3 isoforms of APP mRNA which are regulated through alternative splicing have been reported, along with another minor transcript, APP714 mRNA (Kitaguchi et ai. 1988; Ponte et ai. 1988; Tanzi et al. 1988; Golde et al. 1990). Of these 4 mRNA isoforms, the 2 larger ones (APP770 and 751) encode the Kunitz-type serine protease inhibitor (KPI) domain. It has been reported that mRNA for the protease inhibitor form of APP (APPI) is increased in the AD brain, and that changes in APP mRNA expression may contribute to the deposition of /3/A4 protein (Tanaka et al. 1988, 1989; Johnson et al.
I Present address: Department of Neurology, Kyushu University,
Fukuoka 812, Japan. Correspondence to: Dr. T. Tabira, National Institute of Neuroscience, 4-1-10gawa-higashi, Kodaira, Tokyo 187, Japan, Tel. 42346-1717, Fax 423-46-1747.
1989, 1990). Our previous study showed that neurons abundantly express APP695 mRNA in vitro while astrocytes abundantly express APP751 and APP770 mRNAs (Ohyagi et al. 1990). Moreover, Siman et al. (1989) have reported that reactive astrocytes express APP in vivo following neuronal damage. Therefore, it is possible that the increase of APPI mRNA in the AD brain may be secondary to gliosis and/or neuronal loss, phenomena often found in AD and in aged brains in general. In this study, we used the S1 assay to determine the proportions of APP mRNAs in various cortical samples, and related the results to the histological severity of amyloid deposition, gliosis, and neuronal loss. We found no significant relationship between APP mRNA expression and either amyloid deposition or gliosis/neuronal loss.
Materials and methods
RNA preparation from human organs Postmortem organs were obtained from a non-demented patient (age 50 yr) with spinocerebellar degeneration. Brain tissues were also obtained from 15 autopsy cases at the National Saigata Hospital and the
34 TABLE 1 CLINICAL PROFILES OF THE SUBJECTS Case
Age (yr)
Sex ( F / M )
Clinical diagnosis
Classification
Region
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
63 40 68 57 78 72 80 78 83 78 21 73 87 87 87
M M F M F M M F M M M M F F F
Manic depression Mental retardation OPCA Huntington's disease Cerebral infarct PSP MID + PKD SDAT Mixed dementia Schizophrenia DMD Head trauma SDAT SDAT Cerebral infarct
non-AD non-AD non-AD non-AD non-AD non-AD non-AD AD AD non-AD non-AD AD AD AD Amyloid angiopathy
f f
f f
f f
f f f o o
o o o o
Abbret'iations: AD, Alzheimer's disease; f, frontal cortex; o, occipital cortex; OPCA, olivo-ponto-cerebellar atrophy; PSP, progressive supranuclear palsy; MID, multi-infarct dementia; PKD, Parkinson's disease; SDAT, senile dementia of the Alzheimer type; DMD, Duchenne muscular
dystrophy.
Hizen National Mental Hospital. All tissues were rapidly frozen in liquid nitrogen and stored at -80°C until use. Specific data for each subject are given in Table 1. Total RNA from each tissue was prepared by lysis in 8 M guanidine-HCl, followed by centrifugation through a layer of 5.7 M CsCI (Yamamoto et al. 1983).
template, which had the DdelI-HinclI fragment of human APP770 cDNA containing the KPI domain and a 19-amino acid (19-AA) insert domain ligated at the Hindlll site (Fig, 1). The assay was performed in the presence of an M13 universal primer and Klenow Poll, and with uniform labeling by [a-32p]dCTP. The antisense probe was purified w,ith a 6% (w/v) sequence gel after digestion with EcoRl. Conditions for the assay were essentially similar to those described previously (Barrera-Saldana et al. 1985). After hybridization of 20
SI nuclease protection assay The specific probe for use in the SI nuclease protection assay was synthesized on an M13 mpl8 phage TABLE 2
APP mRNAs COMPARED TO HISTOLOGICAL CHANGES IN THE BRAIN TISSUE EXAMINED Case
1 2 3 4 5 6 7 (Mean 8 9 (Mean 10 11 (Mean 12 13 14 (Mean 15
Proportions of APP mRNAs (%)
Senile plaques
APP695
APP751
APP770
Classical
69.5 51.5 65.0 56.0 68.0 59.9 63.0 61.7 + 66.0 62.0 64.0 + 57 9 58.5 57.7 + 57.0 59.5 61.5 59.3 + 64.0
28.5 42.0 32.0 39.2 30.5 37.0 33.0 34.6 + 31.0 34.0 32.5 + 39.0 37.0 38.0 + 37.5 36.0 33.5 35.7 + 32.0
2.0 6.5 3.0 4.5 1.5 4.0 4.0 3.7 + 1.6) 3.0 4.0 3.5 + 0.5) 4.0 4.5 4.3 + 0.3) 5.5 4.5 5.0 5.0 :t: 0.4) 4.0
. . . +
6.1
2,0
0.8
1.8
4.6
1.5
1.0
1.7
Diffuse . . .
+ + + + + .
Neuronal loss
. . .
Gliosis
. . .
+ +
+ +
+ + + + +
+ + + + + +
+
+ +
+ .
.
_ .
+ + + + +
+ + + + + +
+ :t: + +
+ :1: + +
-
+ +
-
+
1-7, non-AD frontal cortex; 8, 9, AD frontal cortex; 10, 11, non-AD occipital cortex; 12-14, AD occipital cortex; 15, amyloid angiopathy occipital cortex.
35 /tg of total RNA with the probe (5 x 10 4 cpm) overnight at 42°C in 50% formamide, 400 mM NaCI, 40 mM Pipes (pH 6.5) and 1 mM EDTA (pH 7.5), singlestranded DNA was digested with 600 units of S1 nuclease (Takara) for 3 h at 25°C in 30 mM sodium acetate (pH 4.5), 3 mM ZnSO4, and 400 mM NaCI. The sample was then subjected to electrophoresis, and autoradiography. The relative proportions of the 3 APP mRNAs were determined by measuring specific radioactivities with a computer-assisted video image analyser (Fuji BAS2000), and then were standardized by the number of cytosine residues.
+++:+||!
.
meme
-
o
I=LP--+
......
~Z
e
AIq:)770~ ~ o . w . O o
309 APP751.-+ l i l t
Q+OO
1 1 242 238 O 217 qP 201 O 190 e 180
Artificial degradation of RNA
I l l 160
Undegraded total RNA prepared from non-demented human brain tissue was used to examine the effect of RNA degradation on our S1 protection assay. The RNA was hydrolysed in 0.1 M carbonate buffer (40 mM NaHCO 3, 60 mM NaeCO3, pH 10.2) at 65°C for varying lengths of time (3, 10, or 20 min). Hydrolysis was stopped by adding a 1/10 volume 3 M sodium acetate (pH 5.2) and 1/5 volume 10% acetic acid, followed by ethanol precipitation (Golde et ai. 1990). The hydrolysed RNA (10/zg) was then used for Northern blotting and the S1 protection assay.
tip 147
: .
..-
Histological assessment of senile plaques and gliosis/ neuronal loss Classical senile plaques and diffuse plaques were examined by a modified Bielschowsky silver impregnation method (Ogomori et al. 1989). The average plaque density and the average severity of neuronal loss and gliosis were recorded using 4 arbitrary ratings:*-, normal; +, mild; + + , moderate; and + + + , severe (Table 2).
Results APP mRNA expression in systemic organs We analyzed the levels of the 3 APP mRNAs in various human organs in order to determine the speci-
r , ........
=
I-I ..,._.t 89"P
e
e
m
~
(sm~nm)
I l i l l l l l l l l + : , : . . . . | ;';';:;:
I ....... la49
l i
;,,
m ll~ml Kunnzpnmmo Jnhibnor(KJ~)dorian ~9 anmo acid (1OAA)m m dom~
M13 UldVWeidprllnor Fig. l. Diagram of the recombinant phage. The Ddell-Hincll fragment of APP770 eDNA containing the KPI and 19AA insert domains was cloned into the Hindlll site of the MI3 mpl8 phage. After synthesis on this template, the antisense probe was prepared by digestion with EcoRl. The numbering system is according to Kitaguchi et al. (1988).
404
"
41i 122 41; 110
:+
• ,.,++?+..+,
.+,+~" . +
: ; + : ~ 76 12345678 Fig. 2. S1 nuclease protection assay of total RNA from human systemic organs. Numbers on the right side are size markers (nucleotides). Specific bands for each APP mRNA are shown on the left side. FLP = full length probe. Autoradiograph exposure time was 1
day. r J
ficity and sensitivity of our S1 nuclease protection assay system. Since this assay uses an antisense probe derived from human APP770 eDNA, the APP695, 751, and 770 mRNAs should be detected as specific fragments with lengths of 92, 260, and 352 bases, respectively. As shown in Fig. 2, there were multiple bands corresponding to APP695 mRNA. Since all of these bands were less than 92 bases in size, the multiple forms can be explained by variable digestion. APP695 mRNA was detected only in the cerebral and cerebellar cortices. APP751 and 770 mRNAs were detected in all organs in varying amounts, and showed especially high levels in the kidneys and adrenal glands (Fig. 2). Our standard assay system cannot detect an APP?14 mRNA-specific band, because its size is the same as the APP695 mRNA-specific band. Therefore, we tried to distinguish the APPT14 mRNA-specific band by using another shorter probe, but we still could not detect a band for this mRNA (data not shown). APP?51 and 770 mRNAs were found also in the liver, where no APP mRNA was found by Northern blot analysis (data
36 not shown), indicating the higher sensitivity of the S1 assay.
Effect of RNA degradation on the S1 protection assay Before undertaking the analysis of various brain samples by the S1 assay, we had to confirm that the degradation of RNA samples did not affect this assay, because we found moderate degradation of RNA from several brain samples by the Northern blot assay (data not shown). According to the method of Golde et al. (1990), we first prepared various artificially degraded RNA samples. Next, we determined the mean size of the degraded APP mRNA fragments by Northern blotting, perfomed S1 assay of the same samples (Fig. 3A), and obtained the ratio of the 2 results (Fig. 3B). Since the mean size of APP mRNA in our cortical samples
0 3 10 20
0 3 10 20
s
FLP~ APP770-)
M 404
389 APP751--)
242 238 0
APP695--~ [
m,-. emeo APP770"-)
,m * .....
was over 2 kb (data not shown), we confirmed that this assay could determine the precise proportion of APP mRNA even when the samples were-degraded.
APP751-~ ~ ¢ i P
~
, : ~; ;~,~i
8 kb 1.0
4
i0.5 0
H
q w 1 2 3 4 5 6 7 8 9 101112131415
Fig. 4. S1 nuclease protection assay of total RNA from several brains. Size markers and specific bands for each APP mRNA are the same as in Fig. 2. APP695 mRNA-specific bands are indicated on the left side. Case numbers are indicated at the top. Autoradiograph exposure time was 1 day.
A kb
z
3 2 -e
I
I
0
3
I
10 Hydrolysis time
,o|
2O
mln
Fig. 3. (A) Northern blot (left panel) and S1 protection assays (right panel) of artificially hydrolysed human brain RNA. The mean sizes of the degraded APP mRNAs are shown at the left side of the panel. The duration of hydrolysis (rain) is indicated above the panels. (B) Comparison between the APP751/695 mRNA ratio and the mean sizes of degraded APP mRNAs, e, the APP 751/695 mRNA ratio; 8 , the mean sizes of the degraded APP mRNAs.
Analysis of APP mRNAs in Alzheimer and non-Alzheimer brain Subsequently, we determined the proportions of the 3 APP mRNAs in the cerebral cortex in various neurological disorders including Alzheimer's disease (AD). The age of the patients (9 males and 6 females) ranged from 21 to 87 years old (mean age 70 years). We investigated the frontal (A9, 10) or occipital (A17, 18) cortices, because numerous senile plaques were found in these regions by histological examination. The precise region of cortex used was almost the same in every case. According to their clinical features, 3 patients were diagnosed as having senile dementia of the Alzheimer type (Table 1). However, pathological findings such as classical senile plaques led 5 cases in all to be classified as AD after histological examination (Table 2). Although no classical senile plaques were found in case 15, remarkable amyloid angiopathy was found. Mild to moderate gliosis a n d / o r neuronal loss was found in several AD and non-AD brains. The S1 assay data for these cortical tissue samples are shown in Fig. 4. Since we found many minor extra bands above the bands corresponding to APP695 mRNA, the areas indicated on the left side of the panel were used to determine the specific radio activities of APP695 mRNA. The mean APP695 mRNA levels in the frontal and occipital cortices of AD patients were 64% and 59%, respectively, and the levels were slightly higher than
37 TABLE 3 APP mRNAs IN BRAINS WITH NEURONAL LOSS AND GLIOSIS Neuronal loss and gliosis were grouped according to the severity data shown in Table 2. Re-
Grade
n
gion Neuronal loss
f o
Gliosis
f o
Proportion of APP mRNAs (%) APP695
APP751
APP770
-, +, -, +,
+ ++ + ++
6 3 4 2
63.2+6.2 60.3+3.1 59.3+2.6 59.3+2.3
33.5+4.6 33.5+2.9 36.0+2.5 35.5+2.0
3.3+ 1.6 4.2+0.2 4.3+0.3 5.2+0.3
-, +, -, +,
+ ++ + ++
4 5 3 3
63.0+6.8 61.6+4.0 58A+1.0 60.8+2.9
33.4+5.1 34.8+3.1 37.3+ 1.2 34.3+2.3
3.6+ 1.7 3.6+1.1 4.3+0.2 4.9+0.6
n, number of cases; f, frontal cortex; o, occipital cortex.
those for the non-AD brains (62% and 58%, respectively). However, these differences were not significant, because the APP695 mRNA level ranged from 51.5% (Case 2) to 69.5% (Case 1) in AD, exhibiting a relatively large standard deviation (Table 2). In addition, the APP695 mRNA levels in the brains with gliosis/ neuronal loss were slightly lower in the frontal cortex and were the same or slightly higher in the occipital cortex than the levels in the brains without gliosis/ neuronal loss (Table 3). These results may indicate that there is no significant relationship between the proportion of APP mRNA and either amyloid deposition or gliosis/neuronal loss, and suggest that gliosis or neuronal loss does not significantly alter the proportions of these APP mRNAs.
Discussion
The mechanisms of amyloid deposition in AD brains is not yet certain. The hypothesis that alterations in the splicing of APP mRNAs may contribute to amyloid deposition is an interesting one (Tanaka et al. 1988, 1989; Johnson et al. 1989, 1990). According to Tanaka et al. (1989), App751 and 770 mRNA expresssion was significantly increased in AD brains. However, our present study revealed no AD-specific alterations of APP mRNA when compared with other neurodegenerative diseases. Koo et al. (1990) have also reported that APPI mRNA levels do not correlate with amyloid deposition in aged nonhuman primates. Since our assay system is largely the same as the RNase protection assay of Tanaka et aL (1989), we do not know what caused the difference in the results obtained this time. Recently, Golde et al. (1990) determined the proportions of APP mRNAs in precise subregions of the
brain, and found that APP695 mRNA levels were 2-fold higher in the frontal gray matter than in the frontal white matter. They also showed that the increase of APPI mRNA in AD brains was significant in the white matter. Therefore, such regional differences in sampling could perhaps lead to different results. However, since carefully collected similar regions containing mainly gray matter and little white matter from all the brains, the APP mRNA data obtained in this study seem unlikely to have been distorted by such regional differences. Another factor which might affect the proportions of APP mRNAs is an alteration of the APP mRNA-expressing cell population. Previous in situ hybridization studies (Neve et al. 1988; Palmert et al. 1988; Johnson et al. 1990) have revealed that neurons express both APP695 and APPI mRNAs while astrocytes express little APP mRNA compared to neuron in vivo. In contrast, reactive astrocytes express abundant APP following neuronal damage in vivo, but the actual type of APP mRNA involved is still unknown (Siman et al. 1989). In vitro, we have previously shown that neurons express APP695 mRNA while astrocytes express APPI mRNA (Ohyagi et al. 1990). Therefore, it seems more likely that gliosis (reactive astrocytosis) could increase the proportion of APP1 mRNA in the brain. However, our present study suggested that gliosis and/or neuronal loss do not actually alter the proportions of APP mRNAs to a significant degree. This discrepancy could be explained if the expression of APP mRNAs by astrocytes is insufficient in relation to the total amount of gray matter APP mRNA to alter the proportions detected in our assay. Despite the absence of gliosis/ neuronal loss, the APP695 mRNA level was markedly decreased in Case 2. Thus, there is also a possibility that marked alterations of APP mRNA expression may be related to the original disease, and alteration of APP mRNA splicing does not seem to be the cause of amyloid deposition. One of the factors related to amyloid deposition seems to be overexpression of APP. This is supported by the fact that amyloid deposition occurs early in Down syndrome (Wisniewski et ai. 1985), its animal model trisomy 16 mice (Richards et al. 1990), and APP transgenic mice. In APP transgenic mice, Quon et al. (1991) stressed the role of APPI overexpression, but the other group observed amyloid deposition by using the genes encoding the ~-protein portion (Wirak et al. 1991). Therefore, overexpression of the /Lportion of APP seems to be a factor for amyloid deposition. Another factor seems to exist in the processing of APP. Recent studies revealed that APP is concentrated in lysosomes (Benowitz et al. 1989), where APP may be processed by proteolytic enzymes such as cathepsin B (Tagawa et al. 1991), and secreted into the extracellular compartment. Such normal processing
38
cannot generate amyloid protein, because seretase cleaves APP at the mid portion of/~-protein (Esch et al. 1990; Sisodia et al. 1990). However, there exists an evidence showing normal processing generates amyloidogenic carboxyl-terminal derivatives (Estus et al. 1992). Thus, abnormality in the APP processing that generates/3-protein is the most important factor in the amyioidogenesis. Acknowledgements We wish to thank Dr. D. Goldgaber for the generous gift of human APP eDNA, and Miss Y. Kakeba for typing the manuscript. This work was supported in part by gra,~ts from the Japanese Ministryof Health and Welfare (Dementia) and the Health Science Foundation, Japan.
References Barrera-Saldana, H., K. Takahashi, M. Vigneron, A. Wildeman, I. Davidson and P. Chambon (1985) All six GC-motifs of the SV40 early upstream element contribute to promoter activity in vivo and in vitro. EMBO J., 4: 3839-3849. Benowitz, L.i., W. Rodriguez, P. Paskevich, E.J. Mufson, D. Scheuk and R.L. Neve (1989) The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exp. Neurol., 106: 237-250. Esch, F.S., P.S. Keim, E.C. Beattie, R.W. Blacher, A.R. Curwell, T. Oltersdorf, D. McClure and PJ. Ward (1990) Cleavage of amyIoid /3 peptide during constitutive processing of its precursor. Science, 248: 1122-1124. Estus, S., T.E. Golde, T. Kunishita, D. Blades, D. Lowery, M. Eisen, M. Usiak, X. Qu, T. Tabira, B.D. Greenberg and S.G. Younkin (1992) The Alzheimer amyloid protein precursor has potentially amyloidogeniccarboxyl-terminal derivatives. Science. Golde, T.E., S. Estus, M. Usiak, L.H. Younkin and S.G. Younkin (1990) Expression of/3 amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR. Neuron, 4: 253-267. Johnson, S.A., J. Rogers and C.E. Finch (1989) App-695 transcril)t prevalence is selectively reduced during Alzheimer's disease in cortex and hippocampus but not in cerebellum. Neurobiol. Aging, 10: 267-272. Johnson, S.A., T. McNeiU, B. Cordell and C.E. Finch (1990) Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer' s disease. Science, 248: 854-857. Kang, J., H.-G. Lemaire, A. Unterbeck, J.M. Salbaum, C.L. Masters, K.-H. Grzeschik, G. Multhaup, K. Beyreuther and B. Miiller-Hill (1987) The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature, 325: 733-736. Kitaguchi, N., Y. Takahashi, Y. Tokushima, S. Shiojiri and H. Ito (1988) Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature, 331: 530-532. Koo, E.H., S.S. Sisodia, L.C. Cork, A. Unterbeck, R.M. Bayney and D.L. Price (1990) Differential expression of amyloid precursor protein mRNAs in cases of Alzheimer's disease and in aged nonhuman primates. Neuron, 4: 97-104. Neve, R.L., E.A. Finch and L.R. Dawes (1988) Expression of the Alzheimer's amyloid precursor gene transcripts in the human brain. Neuron, h 669-677.
Ogomori, K., T. Kitamoto, J. Tateishi, Y. Satoh, M. Suetsugu and M. Abe (1989) ,B.Protein amyloid is widely distributed in the central nervous system of patients with Alzheimer's disease. Am. J. Pathol., 134: 243-251. Ohyagi, Y., K. Takahashi, M. Kamegai and T. Tabira (1990) Developmental and differential expression of beta amyloid protein precursor mRNAs in mouse brain. Biochem. Biophys. Res. Commun., 167: 54-60. Palmert, M.R., T.E. Golde, M.L. Cohen, D.M. Kovacs, R.E. Tanzi, J.F. Gusella, M.F. Usiak, L.H. Younkin and S.G. Younkin (1988) Amyloid protein precursor messenger RNAs: differential expression in Alzheimer's disease. Science, 241: 1080-1084. Ponte, P., P. Gonzalez-Dewhitt, J. Schilling, J. Miller, D. Hsu, B. Greenberg, K. Davis, W. Wallence, !. Lieberburg, F. Fuller and B. Cordell (1988) A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature, 331: 525-527. Quon, D., Y. Wang, R. Catalano, J.M. Scardina, K. Murakami and B. Cordell (1991) Formation of /3-amyloid protein deposits in brains of transgenic mice. Nature, 352: 239-241. Richards, S.-J., J.J. Waters, D.C. Rogers, F.L. Martel, D.R. Sparkman, C.L. White, K. Beyreuther, C.L. Masters and S.B. Dunnett (1990) Hippocampal grafts derived from embryonic trisomy 16 mice exhibit amyloid (A4) and neurofibrillary pathology. In: S.B. Dunnett and S.-J. Richards (Eds.), Neural Transplantation: From Molecular Basis to Clinical Applications, Progress in Brain Research, Vol. 82, Elsevier, Amsterdam, pp. 215-223. Siman, R., J.P. Card, R.B. Nelson and L.G. Davis (1989) Expression of /3-amyloid precursor protein in reactive astrocytes following neuronal damage. Neuron, 3: 275-285. Sisodia, S.S., E.H. Koo, IL Beyreuther, A. Unterbeck and D.L. Price (1990) Evidence that/3-amyloid protein in.Alzheimer's disease is not derived by normal processing. Science, 248: 492-495. Tagawa, K., T. Kunishita, K. Maruyama, K. Yoshikawa, E. Kominami, T. Tsuchiya, K. Suzuki, T. Tabira, H. Sugita and S. lshiura (1991) Alzheimer's disease amyloid ,8-clipping enzyme (APP secretase): identification, purification, and characterization of the enzyme. Biochem. Biophys. Res. Commun., 177: 377-387. Tanaka, S., S. Nakayama, K. Ueda, M. Kameyama, S. Shiojiri, Y. Takahashi, N. Kitaguchi and H. ito (1988) Three types of amyloid protein precursor mRNA in human brain; their differential expression in Alzheimer's disease. Biochem. Biophys. Res. Cornmen., 157: 472-479. Tanaka, S., S. Shiojiri, Y. Takahashi, N. Kitaguchi, H. lto, M. Kameyama, J. Kimura, S. Nakamura and K. Ueda (1989) Tissuespecific expression of three types of/3-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain. Biochem. Biophys. Res. Commun., 165: 1406-1414. Tanzi, R.E., A.I. McClatchey, E.D. Lamperti, L. Villa-Komaroff,J.F. Gusella and R.L. Neve (1988) Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease. Nature, 331: 528-530. -~ Wirak, D.O., R. Bayney, T.V. Ramabhadran, R.P. Francasso, J.T. Hart, P.E. Hauer, P. Hsiau, S.K. Pekar, G.A. Scangos, B.D. Trapp and A.J. Unterbeck (1991) Deposits of amyloid/J protein in the central nervous system of transgenic mice. Science, 253: 323-325. Wisniewski, K.E., A.J. Dalton, D.R.C. McLachlan, G.Y. Wen and H.M. Wisniewski (1985) Alzheimer's disease in Down's syndrome: clinicopathologic studies. Neurology, 35: 957-961. Yamamoto, M., Y. Maehara, K. Takahashi and H. Endo (1983) Cloning of sequences expressed specifically in tumors of rat, Pro(:. Natl. Acad. Sci. USA, 80: 7524-7527.
33
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00 JNS 03800
Cerebral cortical amyloid protein precursor mRNA expression is similar in Alzheimer's disease and other neurodegenerative diseases Yasumasa Ohyagi
a,1, Keikichi T a k a h a s h i
a, Y u h j i S a t o h b, T a k a o M a k i f u c h i c and Takeshi Tabira a
a Division of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan, b Hizen National Mental Hospital, Kanzaki, Saga, Japan, and c National Saigata Hospital, Ogata-town, Niigata, Japan
(Received 6 December, 1991) (Revised, received 16 February, 1992) (Accepted 15 February, 1992) Key words: Amyloid protein precursor mRNA; Alzheimer's disease; Neurodegenerative disease; Gliosis; Neuronal loss
Summary The expression of 3/3-amyloid protein precursor (APP) mRNAs (695, 751, and 770) in the cerebral cortex in Alzheimer's disease and other neurodegenerative diseases was analyzed by the SI nuclease protection assay. We found no significant Alzheimer's disease-specific alteration of APP mRNA expression when compared to the other neurological diseases as controls. Since the expression of this mRNA was not correlated with amyloid deposition, it is possible that gliosis/neuronal loss may secondarily alter APP mRNA expression. However, the current study revealed no significant correlation between them.
Introduction
In the Alzheimer's disease (AD) brain, large numbers of senile plaques are found throughout the neocortex and hippocampus. Beta (or A4)-protein is the major component of senile plaque amyloid and is thought to be derived from amyloid protein precursors (APP) (Kang et al. 1987). At least 3 isoforms of APP mRNA which are regulated through alternative splicing have been reported, along with another minor transcript, APP714 mRNA (Kitaguchi et ai. 1988; Ponte et ai. 1988; Tanzi et al. 1988; Golde et al. 1990). Of these 4 mRNA isoforms, the 2 larger ones (APP770 and 751) encode the Kunitz-type serine protease inhibitor (KPI) domain. It has been reported that mRNA for the protease inhibitor form of APP (APPI) is increased in the AD brain, and that changes in APP mRNA expression may contribute to the deposition of /3/A4 protein (Tanaka et al. 1988, 1989; Johnson et al.
I Present address: Department of Neurology, Kyushu University,
Fukuoka 812, Japan. Correspondence to: Dr. T. Tabira, National Institute of Neuroscience, 4-1-10gawa-higashi, Kodaira, Tokyo 187, Japan, Tel. 42346-1717, Fax 423-46-1747.
1989, 1990). Our previous study showed that neurons abundantly express APP695 mRNA in vitro while astrocytes abundantly express APP751 and APP770 mRNAs (Ohyagi et al. 1990). Moreover, Siman et al. (1989) have reported that reactive astrocytes express APP in vivo following neuronal damage. Therefore, it is possible that the increase of APPI mRNA in the AD brain may be secondary to gliosis and/or neuronal loss, phenomena often found in AD and in aged brains in general. In this study, we used the S1 assay to determine the proportions of APP mRNAs in various cortical samples, and related the results to the histological severity of amyloid deposition, gliosis, and neuronal loss. We found no significant relationship between APP mRNA expression and either amyloid deposition or gliosis/neuronal loss.
Materials and methods
RNA preparation from human organs Postmortem organs were obtained from a non-demented patient (age 50 yr) with spinocerebellar degeneration. Brain tissues were also obtained from 15 autopsy cases at the National Saigata Hospital and the
34 TABLE 1 CLINICAL PROFILES OF THE SUBJECTS Case
Age (yr)
Sex ( F / M )
Clinical diagnosis
Classification
Region
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
63 40 68 57 78 72 80 78 83 78 21 73 87 87 87
M M F M F M M F M M M M F F F
Manic depression Mental retardation OPCA Huntington's disease Cerebral infarct PSP MID + PKD SDAT Mixed dementia Schizophrenia DMD Head trauma SDAT SDAT Cerebral infarct
non-AD non-AD non-AD non-AD non-AD non-AD non-AD AD AD non-AD non-AD AD AD AD Amyloid angiopathy
f f
f f
f f
f f f o o
o o o o
Abbret'iations: AD, Alzheimer's disease; f, frontal cortex; o, occipital cortex; OPCA, olivo-ponto-cerebellar atrophy; PSP, progressive supranuclear palsy; MID, multi-infarct dementia; PKD, Parkinson's disease; SDAT, senile dementia of the Alzheimer type; DMD, Duchenne muscular
dystrophy.
Hizen National Mental Hospital. All tissues were rapidly frozen in liquid nitrogen and stored at -80°C until use. Specific data for each subject are given in Table 1. Total RNA from each tissue was prepared by lysis in 8 M guanidine-HCl, followed by centrifugation through a layer of 5.7 M CsCI (Yamamoto et al. 1983).
template, which had the DdelI-HinclI fragment of human APP770 cDNA containing the KPI domain and a 19-amino acid (19-AA) insert domain ligated at the Hindlll site (Fig, 1). The assay was performed in the presence of an M13 universal primer and Klenow Poll, and with uniform labeling by [a-32p]dCTP. The antisense probe was purified w,ith a 6% (w/v) sequence gel after digestion with EcoRl. Conditions for the assay were essentially similar to those described previously (Barrera-Saldana et al. 1985). After hybridization of 20
SI nuclease protection assay The specific probe for use in the SI nuclease protection assay was synthesized on an M13 mpl8 phage TABLE 2
APP mRNAs COMPARED TO HISTOLOGICAL CHANGES IN THE BRAIN TISSUE EXAMINED Case
1 2 3 4 5 6 7 (Mean 8 9 (Mean 10 11 (Mean 12 13 14 (Mean 15
Proportions of APP mRNAs (%)
Senile plaques
APP695
APP751
APP770
Classical
69.5 51.5 65.0 56.0 68.0 59.9 63.0 61.7 + 66.0 62.0 64.0 + 57 9 58.5 57.7 + 57.0 59.5 61.5 59.3 + 64.0
28.5 42.0 32.0 39.2 30.5 37.0 33.0 34.6 + 31.0 34.0 32.5 + 39.0 37.0 38.0 + 37.5 36.0 33.5 35.7 + 32.0
2.0 6.5 3.0 4.5 1.5 4.0 4.0 3.7 + 1.6) 3.0 4.0 3.5 + 0.5) 4.0 4.5 4.3 + 0.3) 5.5 4.5 5.0 5.0 :t: 0.4) 4.0
. . . +
6.1
2,0
0.8
1.8
4.6
1.5
1.0
1.7
Diffuse . . .
+ + + + + .
Neuronal loss
. . .
Gliosis
. . .
+ +
+ +
+ + + + +
+ + + + + +
+
+ +
+ .
.
_ .
+ + + + +
+ + + + + +
+ :t: + +
+ :1: + +
-
+ +
-
+
1-7, non-AD frontal cortex; 8, 9, AD frontal cortex; 10, 11, non-AD occipital cortex; 12-14, AD occipital cortex; 15, amyloid angiopathy occipital cortex.
35 /tg of total RNA with the probe (5 x 10 4 cpm) overnight at 42°C in 50% formamide, 400 mM NaCI, 40 mM Pipes (pH 6.5) and 1 mM EDTA (pH 7.5), singlestranded DNA was digested with 600 units of S1 nuclease (Takara) for 3 h at 25°C in 30 mM sodium acetate (pH 4.5), 3 mM ZnSO4, and 400 mM NaCI. The sample was then subjected to electrophoresis, and autoradiography. The relative proportions of the 3 APP mRNAs were determined by measuring specific radioactivities with a computer-assisted video image analyser (Fuji BAS2000), and then were standardized by the number of cytosine residues.
+++:+||!
.
meme
-
o
I=LP--+
......
~Z
e
AIq:)770~ ~ o . w . O o
309 APP751.-+ l i l t
Q+OO
1 1 242 238 O 217 qP 201 O 190 e 180
Artificial degradation of RNA
I l l 160
Undegraded total RNA prepared from non-demented human brain tissue was used to examine the effect of RNA degradation on our S1 protection assay. The RNA was hydrolysed in 0.1 M carbonate buffer (40 mM NaHCO 3, 60 mM NaeCO3, pH 10.2) at 65°C for varying lengths of time (3, 10, or 20 min). Hydrolysis was stopped by adding a 1/10 volume 3 M sodium acetate (pH 5.2) and 1/5 volume 10% acetic acid, followed by ethanol precipitation (Golde et ai. 1990). The hydrolysed RNA (10/zg) was then used for Northern blotting and the S1 protection assay.
tip 147
: .
..-
Histological assessment of senile plaques and gliosis/ neuronal loss Classical senile plaques and diffuse plaques were examined by a modified Bielschowsky silver impregnation method (Ogomori et al. 1989). The average plaque density and the average severity of neuronal loss and gliosis were recorded using 4 arbitrary ratings:*-, normal; +, mild; + + , moderate; and + + + , severe (Table 2).
Results APP mRNA expression in systemic organs We analyzed the levels of the 3 APP mRNAs in various human organs in order to determine the speci-
r , ........
=
I-I ..,._.t 89"P
e
e
m
~
(sm~nm)
I l i l l l l l l l l + : , : . . . . | ;';';:;:
I ....... la49
l i
;,,
m ll~ml Kunnzpnmmo Jnhibnor(KJ~)dorian ~9 anmo acid (1OAA)m m dom~
M13 UldVWeidprllnor Fig. l. Diagram of the recombinant phage. The Ddell-Hincll fragment of APP770 eDNA containing the KPI and 19AA insert domains was cloned into the Hindlll site of the MI3 mpl8 phage. After synthesis on this template, the antisense probe was prepared by digestion with EcoRl. The numbering system is according to Kitaguchi et al. (1988).
404
"
41i 122 41; 110
:+
• ,.,++?+..+,
.+,+~" . +
: ; + : ~ 76 12345678 Fig. 2. S1 nuclease protection assay of total RNA from human systemic organs. Numbers on the right side are size markers (nucleotides). Specific bands for each APP mRNA are shown on the left side. FLP = full length probe. Autoradiograph exposure time was 1
day. r J
ficity and sensitivity of our S1 nuclease protection assay system. Since this assay uses an antisense probe derived from human APP770 eDNA, the APP695, 751, and 770 mRNAs should be detected as specific fragments with lengths of 92, 260, and 352 bases, respectively. As shown in Fig. 2, there were multiple bands corresponding to APP695 mRNA. Since all of these bands were less than 92 bases in size, the multiple forms can be explained by variable digestion. APP695 mRNA was detected only in the cerebral and cerebellar cortices. APP751 and 770 mRNAs were detected in all organs in varying amounts, and showed especially high levels in the kidneys and adrenal glands (Fig. 2). Our standard assay system cannot detect an APP?14 mRNA-specific band, because its size is the same as the APP695 mRNA-specific band. Therefore, we tried to distinguish the APPT14 mRNA-specific band by using another shorter probe, but we still could not detect a band for this mRNA (data not shown). APP?51 and 770 mRNAs were found also in the liver, where no APP mRNA was found by Northern blot analysis (data
36 not shown), indicating the higher sensitivity of the S1 assay.
Effect of RNA degradation on the S1 protection assay Before undertaking the analysis of various brain samples by the S1 assay, we had to confirm that the degradation of RNA samples did not affect this assay, because we found moderate degradation of RNA from several brain samples by the Northern blot assay (data not shown). According to the method of Golde et al. (1990), we first prepared various artificially degraded RNA samples. Next, we determined the mean size of the degraded APP mRNA fragments by Northern blotting, perfomed S1 assay of the same samples (Fig. 3A), and obtained the ratio of the 2 results (Fig. 3B). Since the mean size of APP mRNA in our cortical samples
0 3 10 20
0 3 10 20
s
FLP~ APP770-)
M 404
389 APP751--)
242 238 0
APP695--~ [
m,-. emeo APP770"-)
,m * .....
was over 2 kb (data not shown), we confirmed that this assay could determine the precise proportion of APP mRNA even when the samples were-degraded.
APP751-~ ~ ¢ i P
~
, : ~; ;~,~i
8 kb 1.0
4
i0.5 0
H
q w 1 2 3 4 5 6 7 8 9 101112131415
Fig. 4. S1 nuclease protection assay of total RNA from several brains. Size markers and specific bands for each APP mRNA are the same as in Fig. 2. APP695 mRNA-specific bands are indicated on the left side. Case numbers are indicated at the top. Autoradiograph exposure time was 1 day.
A kb
z
3 2 -e
I
I
0
3
I
10 Hydrolysis time
,o|
2O
mln
Fig. 3. (A) Northern blot (left panel) and S1 protection assays (right panel) of artificially hydrolysed human brain RNA. The mean sizes of the degraded APP mRNAs are shown at the left side of the panel. The duration of hydrolysis (rain) is indicated above the panels. (B) Comparison between the APP751/695 mRNA ratio and the mean sizes of degraded APP mRNAs, e, the APP 751/695 mRNA ratio; 8 , the mean sizes of the degraded APP mRNAs.
Analysis of APP mRNAs in Alzheimer and non-Alzheimer brain Subsequently, we determined the proportions of the 3 APP mRNAs in the cerebral cortex in various neurological disorders including Alzheimer's disease (AD). The age of the patients (9 males and 6 females) ranged from 21 to 87 years old (mean age 70 years). We investigated the frontal (A9, 10) or occipital (A17, 18) cortices, because numerous senile plaques were found in these regions by histological examination. The precise region of cortex used was almost the same in every case. According to their clinical features, 3 patients were diagnosed as having senile dementia of the Alzheimer type (Table 1). However, pathological findings such as classical senile plaques led 5 cases in all to be classified as AD after histological examination (Table 2). Although no classical senile plaques were found in case 15, remarkable amyloid angiopathy was found. Mild to moderate gliosis a n d / o r neuronal loss was found in several AD and non-AD brains. The S1 assay data for these cortical tissue samples are shown in Fig. 4. Since we found many minor extra bands above the bands corresponding to APP695 mRNA, the areas indicated on the left side of the panel were used to determine the specific radio activities of APP695 mRNA. The mean APP695 mRNA levels in the frontal and occipital cortices of AD patients were 64% and 59%, respectively, and the levels were slightly higher than
37 TABLE 3 APP mRNAs IN BRAINS WITH NEURONAL LOSS AND GLIOSIS Neuronal loss and gliosis were grouped according to the severity data shown in Table 2. Re-
Grade
n
gion Neuronal loss
f o
Gliosis
f o
Proportion of APP mRNAs (%) APP695
APP751
APP770
-, +, -, +,
+ ++ + ++
6 3 4 2
63.2+6.2 60.3+3.1 59.3+2.6 59.3+2.3
33.5+4.6 33.5+2.9 36.0+2.5 35.5+2.0
3.3+ 1.6 4.2+0.2 4.3+0.3 5.2+0.3
-, +, -, +,
+ ++ + ++
4 5 3 3
63.0+6.8 61.6+4.0 58A+1.0 60.8+2.9
33.4+5.1 34.8+3.1 37.3+ 1.2 34.3+2.3
3.6+ 1.7 3.6+1.1 4.3+0.2 4.9+0.6
n, number of cases; f, frontal cortex; o, occipital cortex.
those for the non-AD brains (62% and 58%, respectively). However, these differences were not significant, because the APP695 mRNA level ranged from 51.5% (Case 2) to 69.5% (Case 1) in AD, exhibiting a relatively large standard deviation (Table 2). In addition, the APP695 mRNA levels in the brains with gliosis/ neuronal loss were slightly lower in the frontal cortex and were the same or slightly higher in the occipital cortex than the levels in the brains without gliosis/ neuronal loss (Table 3). These results may indicate that there is no significant relationship between the proportion of APP mRNA and either amyloid deposition or gliosis/neuronal loss, and suggest that gliosis or neuronal loss does not significantly alter the proportions of these APP mRNAs.
Discussion
The mechanisms of amyloid deposition in AD brains is not yet certain. The hypothesis that alterations in the splicing of APP mRNAs may contribute to amyloid deposition is an interesting one (Tanaka et al. 1988, 1989; Johnson et al. 1989, 1990). According to Tanaka et al. (1989), App751 and 770 mRNA expresssion was significantly increased in AD brains. However, our present study revealed no AD-specific alterations of APP mRNA when compared with other neurodegenerative diseases. Koo et al. (1990) have also reported that APPI mRNA levels do not correlate with amyloid deposition in aged nonhuman primates. Since our assay system is largely the same as the RNase protection assay of Tanaka et aL (1989), we do not know what caused the difference in the results obtained this time. Recently, Golde et al. (1990) determined the proportions of APP mRNAs in precise subregions of the
brain, and found that APP695 mRNA levels were 2-fold higher in the frontal gray matter than in the frontal white matter. They also showed that the increase of APPI mRNA in AD brains was significant in the white matter. Therefore, such regional differences in sampling could perhaps lead to different results. However, since carefully collected similar regions containing mainly gray matter and little white matter from all the brains, the APP mRNA data obtained in this study seem unlikely to have been distorted by such regional differences. Another factor which might affect the proportions of APP mRNAs is an alteration of the APP mRNA-expressing cell population. Previous in situ hybridization studies (Neve et al. 1988; Palmert et al. 1988; Johnson et al. 1990) have revealed that neurons express both APP695 and APPI mRNAs while astrocytes express little APP mRNA compared to neuron in vivo. In contrast, reactive astrocytes express abundant APP following neuronal damage in vivo, but the actual type of APP mRNA involved is still unknown (Siman et al. 1989). In vitro, we have previously shown that neurons express APP695 mRNA while astrocytes express APPI mRNA (Ohyagi et al. 1990). Therefore, it seems more likely that gliosis (reactive astrocytosis) could increase the proportion of APP1 mRNA in the brain. However, our present study suggested that gliosis and/or neuronal loss do not actually alter the proportions of APP mRNAs to a significant degree. This discrepancy could be explained if the expression of APP mRNAs by astrocytes is insufficient in relation to the total amount of gray matter APP mRNA to alter the proportions detected in our assay. Despite the absence of gliosis/ neuronal loss, the APP695 mRNA level was markedly decreased in Case 2. Thus, there is also a possibility that marked alterations of APP mRNA expression may be related to the original disease, and alteration of APP mRNA splicing does not seem to be the cause of amyloid deposition. One of the factors related to amyloid deposition seems to be overexpression of APP. This is supported by the fact that amyloid deposition occurs early in Down syndrome (Wisniewski et ai. 1985), its animal model trisomy 16 mice (Richards et al. 1990), and APP transgenic mice. In APP transgenic mice, Quon et al. (1991) stressed the role of APPI overexpression, but the other group observed amyloid deposition by using the genes encoding the ~-protein portion (Wirak et al. 1991). Therefore, overexpression of the /Lportion of APP seems to be a factor for amyloid deposition. Another factor seems to exist in the processing of APP. Recent studies revealed that APP is concentrated in lysosomes (Benowitz et al. 1989), where APP may be processed by proteolytic enzymes such as cathepsin B (Tagawa et al. 1991), and secreted into the extracellular compartment. Such normal processing
38
cannot generate amyloid protein, because seretase cleaves APP at the mid portion of/~-protein (Esch et al. 1990; Sisodia et al. 1990). However, there exists an evidence showing normal processing generates amyloidogenic carboxyl-terminal derivatives (Estus et al. 1992). Thus, abnormality in the APP processing that generates/3-protein is the most important factor in the amyioidogenesis. Acknowledgements We wish to thank Dr. D. Goldgaber for the generous gift of human APP eDNA, and Miss Y. Kakeba for typing the manuscript. This work was supported in part by gra,~ts from the Japanese Ministryof Health and Welfare (Dementia) and the Health Science Foundation, Japan.
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