Molecular Brain Research, 15 (1992) 303-310
303
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70486
Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimer's disease and other neurological disorders Seigo Tanaka
a
Li Liu
b
Jun Kimura
b
S a t o s h i S h i o j i r i c, Y a s u y u k i T a k a h a s h i
N o b u y a K i t a g u c h i c, S h i g e n o b u N a k a m u r a
d and Kunihiro Ueda
c,
a
a Department of Clinical Science and Laboratory Medicine, b Department of Neurology, Kyoto University Faculty of Medicine, Kyoto (Japan), c Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry, Shizuoka (Japan) and d 3rd Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan)
(Accepted 19 May 1992)
Key words: Alzheimer'sdisease;/3A4 amyloid;Amyloidprecursor protein; Alternative splicing; Protease inhibitor; Aging;
RNase protection assay
In the human brain, alternative splicing of amyloid precursor protein (APP) gene transcript generates at least three types of mRNA coding for APP770, APP751 and APP695.The former two types harbor, but the latter'one lacks a domain of Kunitz-type serine protease inhibitor (KPI). We studied, by using the RNase protection technique, the expression of APP mRNAs in brains of Alzheimer's disease (AD) and other neurological disorders with special reference to aging. We found that the ratio of (APP770 mRNA+APP751 mRNA)/APP695 mRNA in the frontal cortex increased approximately 1.5-fold in AD compared with other neurodegenerative or cerebrovascular disorders. The ratio in other neurological disorders did not change significantly from control even in their affected brain regions. On the other hand, we found a positive correlation between the ratio and age; the ratio (y) increased gradually with the advance of age (x) as expressed by y = 0.005x +0.014 (r = 0.372) for the AD group, and y = 0.004x -0.037 (r = 0.486) for the non-AD group. These correlations indicate that the AD brain reached the same ratio of KPl-harboring to lacking APP mRNAs a few decades earlier than the non-AD brain in senescence. This finding of AD-specificand age-related change led us to the idea that a relative increase in KPI-harboringAPPs over a KPI-lacking APP may perturb normal degradation of APPs, thereby leading to deposition of/3A4 protein as amyloid.
INTRODUCTION Alzheimer's disease (AD) constitutes one of the most common neurodegenerative disorders, presenting as progressive dementia with loss of memory, intellectual disturbance, and language problems. The pathogenesis of this disease, although unknown, evolves around the deposition o f / 3 A 4 protein in senile plaque cores and cerebral vessels 6'22'23'26'45.The flA4 protein is generated from larger precursors (amyloid precursor proteins; APPs) that have structual features of cell surface receptors ~3. Three (or reportedly four) types of APP m R N A [APP770, APP751, (APPTt 4) and APP695 mRNAs] are produced from a single gene transcript by alternative splicing of exon 7 and 8 segments 2°'4s. The 56 amino acid sequence encoded by exon 7 is highly homologous to a Kunitz-type serine protease inhibitor
(KPI)14'34'42 and
has a protease inhibitor activity, as demonstrated by an in vitro expression experiment 14. This KPI segment is harbored in APP770 and APP751, but not in APP695 (nor APP714). Our previous study 41 showed that the proportion of APP770 m R N A (or APP770 m R N A + APP751 m R N A ) in various brain regions is higher in A D than in control. The highest level was observed in the cerebral cortex and hippocampus known to have the highest density of senile plaques. In addition, A D samples showing histologically a high density of senile plaques exhibited a high ratio of (APP770 m R N A + APP751 m R N A ) / A P P 6 9 5 m R N A 1~'41. These results led us to an inference that an increased expression of KPI-harboring APPs relative to KPI-lacking one(s) might affect normal degradation of APPs in the brain and lead to accumulation of aberrant proteolytic products as amyloid.
Correspondence: S. Tanaka, Department of Clinical Science and Laboratory Medicine, KyotoUniversity Faculty of Medicine, Shogoin, Sakyo-ku,
Kyoto 606, Japan. Fax: (81) 75-771-4792.
304 Neurodegenerative changes are observed in Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and spinocerebellar degeneration (SCD), in addition to AD. These diseases share, more or less, common pathological changes such as a gradual decay and loss of neurons accompanied by gliosis at specific brain regions 3°. Senile plaques increase with the advance of age in patients with these disorders as well as persons with no neurological disorder, although to a much lesser degree than in AD patients. In view of a good correlation between the density of senile plaques and APP mRNAs proportion ~2'4t, we addressed the following questions in the present study: (1) Do A P P 7 7 0 and APP751 mRNAs increase in other neurological disorders? (2) Does the APP mRNAs proportion change with aging? And (3) does the APP mRNAs proportion differ between the AD group and the non-AD group in an age-matched comparison? Our results indicate that AD exhibits a unique change in the APP mRNAs expression, not shared by other neurological disorders until far later senescence, in the brain. MATERIALS A N D METHODS Brain samples and total RNA isolation Postmortem brain samples were obtained, within 12 h after death, from patients with AD, mixed-type dementia, multi-infarct dementia (MID), PD, ALS, SCD ( O P C A type), cerebral infarction and cerebral hemorrhage, and persons without neurological disorders (control) (Table IA). O n e half of the brain, frozen at - 8 0 ° C , was used for m R N A analysis, and the other half, fixed in formalin, for histological examination. We diagnosed A D clinically using the N I N C D S - A D R D A criteria 24 and histologically by the density of
senile plaques and neurofibrillary tangles stained with the silver impregnation method. MID patients showed histologically multiple cerebral infarction with atherosclerosis and arteriolar sclerosis as underlying vascular lesions. Mixed-type dementia is a state exhibiting both AD-type changes and vascular lesions. We made diagnosis of other disorders clinically and confirmed the absence of AD-type changes by histological examination. The midbrain of PD patients showed loss of substantia nigral cells and gliosis with Lewy body formation. The motor area (Brodmann area 4) of two patients with ALS showed mild loss of giant pyramidal cells of Betz. The cerebellar cortex of SCD patient showed loss of Purkinje cells. We cut out the frontal cortex (Brodmann areas 9 and 10) from the frozen brain of all patients, and also specific brain regions affected in each neurodegenerative disorder mentioned above. Sample of patients with cerebral infarction or hemorrhage was from the region devoid of major lesions. We obtained sample also from the whole brain of fetus aborted at 10 weeks. Total cellular R N A was extracted from brain samples by the g u a n i d i n i u m / C s C I method 3.
RNase protection assay The antisense R N A probe for RNase protection assay 25 (Fig. 1) was prepared as follows. A Ddel (nt 7 7 4 ) - X h o i (nt 1136)fragment of APP770 c D N A 14 encompassing exons 7 and 8 was inserted inversely into pSP64 plasmid (Promega) downstream to the SP6 promotor site, and the plasmid was proliferated in Escherichia coli. Using the EcoR1 fragment of the plasmid D N A (1 /xg) as template, antisense R N A fragment was synthesized at 40°C for 30 min with 5 units of SP6 R N A polymerase (BRL) in 10 txl of the reaction mixture [40 m M Tris-Cl (pH 7.5), 6 m M MgCI2, 2 m M spermidine, 0.5 m M each ATP, U T P and GTP, and 12.5 ~ M [a- 32p]CTP (400 Ci/mmol)]. The mixture was then supplemented with 1 /~g of RNase-free DNase I (BRL), and further incubated at 37°C for 10 rain. R N A was extracted from the mixture with phenol/chloroform, and precipitated with ethanol. The R N A probe thus 32p-labelled (5 × 105 cpm) was hybridized with sample R N A (10 ~tg) in 3 0 / z l of the hybridization buffer [80% formamide, 40 m M PIPES (pH 6.4), 400 m M NaCI, and 1 m M EDTA] at 45°C for 12 h. The hybridization mixture was treated at 25°C for 10 min with RNase A (Sigma; 40 t z g / m l ) and RNase T1 (BRL; 2 ~zg/ml) in 300 tzl of the digestion buffer [10 m M Tris-Cl (pH 7.5), 300 m M NaCI, and 5 m M EDTA]. After addition of 20 tzl of 10% SDS and 10 #1 of proteinase K (Merck; 5 m g / m l ) to
TABLE 1
APP mRNAs proportion in the frontal cortex of t:arious disorders * Proportions were calculated from radioactivities of band areas located by autoradiography (Fig. 2) as described in Materials and Methods. ** Samples obtained from persons in seventies and eighties of age were compared. The A D group includes A D and mixed-type dementia. Values are mean -+ S.D.
Disorder or group
n
Age (years)
mRNA proportion * (%)
Ratio
APP77o
APP751
App~o5
~a)
(b)
(c)
3.5_+ 1.9 2.2 _+0.5 2.4 _+ 1.6 1.9-+ 1.0 2.6 _+ 1.7 1.1 1.7 _+0.8 a 1.4 _+0.8 1.7 + 0.7 a
25.2_+5.3 23.2 _+4.7 22.4 _+4.9 18.8-+4.8 19.4 _+6.2 22.8 20.6 _+6.4 20.0 _+6.1 19.0 + 5.6
71.3_+6.9 74.7 _+4.2 75.2 _+5.8 79.3_+5.7 78.0 _+7.8 76.1 77.7 _+6.0 78.5 _+5.9 79.3 + 6.2 "
0.41 _+0.12 0.34 _+0.07 0.34 ± 0.11 0.27_+0.09 ~' 0.30 5:0.13 0.31 0.29 5:0.09 0.28 5:0.09 0.27 ± 0.11 a
B. Age-matched comparison between A D and non-AD groups ** A D group 8 78.9_+ 5.2 3.2_+2.0 N o n - A D group 21 77.9_+ 5.1 1.7_+ 1.1 b
24.3_+5.4 20.6_+4.8
72.4_+7.(I 77.7_+5.1 b
0.39_+0.13 0.29_+0.08 b
A. Comparison among neurological disorders AD 8 74.8_+ 9.2 Mixed-type dementia 2 79.0 _+ 7.1 MID 6 81.5 _+ 11.1 PD 5 70.6-+ 2.7 ALS 4 65.0 _+ 7.4 SCD 1 57.0 Infarction 6 78.2 -+ 4.7 Hemorrhage 4 85.5 -+ 6.6 Control 7 75.3 -+ 13.5
P < 0.05 (Student's t-test), compared with AD; b p < 0.05, compared with A D group.
(a + b) / c
305 regression method to analyze correlation between the ratio and age. The results were expressed as a regression line accompanied by 90% confidence lines and a correlation coefficient (r).
KPI segment !
APP gene transcript
RESULTS
APP,,0
l/ I 0
Proportion o f A P P m R N A s in the brain
367nt
T h e R N a s e p r o t e c t i o n assay of b r a i n A P P m R N A s revealed four b a n d s in a u t o r a d i o g r a p h y at the position
261nt APPT14 mRNA
/
~
m R N A s e n c o d i n g APP770, APP751, APP69s, a n d APP7s 1 plus APP695, respectively (Fig. 1); the 93 n t b a n d was
/ 93 nt
APP695 mRNA
of 367, 261, 93 a n d 49 nt long (Fig. 2), c o r r e s p o n d i n g to
49nt
/~r'~-]~_ 93nt
i n t e r p r e t e d to r e p r e s e n t solely APP69 s m R N A , b e c a u s e
106 nt
/ 49nt
Fig. 1. Structures of APP gene transcript and four types of spliced APP mRNAs in relation to the sites hybridizing with antisense RNA probe in the RNase protection assay. The number of each domain corresponds to that of exon in the APP gene. Exon 7 encodes the KPI segment. Solid bars accompanied by lengths in nucleotides (nt) represent the fragments of the probe protected from RNase digestion.
the solution, the mixture was incubated at 37°C for 15 min. RNA was extracted with phenol/chloroform and precipitated with ethanol, and the pellet was dissolved in the loading buffer [80% formamide, 1 mM EDTA (pH 8.0), 0.1% bromophenol blue, and 0.1% xylene cyanol]. RNA hybrids were denatured, and electrophoresed on 6% polyacrylamide/10 M urea gel. Finally, we subjected the gel to autoradiography using an intensifying screen and Kodak X-Omat film. For quantitative analysis, band areas located by autoradiography were cut out from dried gel, and their radioactivities were determined by the liquid scintillation method. The values thus obtained were corrected for the number of cytosine residues contained in each fragment for calculation of APP mRNAs proportion.
Statistical analysis We used Student's t-test to evaluate significance of a difference in the ratio of APP mRNAs between two groups, and the linear
a 106 n t b a n d expected for APP714 m R N A was u n d e tectable u n d e r o u r e x p e r i m e n t a l conditions. A m o n g the four b a n d s , the 367 nt b a n d was the least promin e n t a n d hardly d e t e c t a b l e in some samples unless a l o n g e r exposure of the gel. O t h e r m i n o r b a n d s emerging variably r e m a i n to be characterized. Q u a n t i t a t i v e analysis of radioactivities revealed that APP695 m R N A a b o u n d e d in the brain, followed by APP75 t m R N A (Table IA). APP770 m R N A was a m i n o r c o m p o n e n t , regardless of b r a i n regions.
Difference in the A P P m R N A s proportion a m o n g neurological disorders W e investigated the difference in the A P P m R N A s p r o p o r t i o n a m o n g various n e u r o l o g i c a l disorders, first, in an identical b r a i n region, i.e., the frontal cortex (Fig. 2 A - I ) . As a distinct finding, the a u t o r a d i o g r a m s of several A D samples exhibited a d e n s e 367 nt b a n d (APP770 m R N A ) . Q u a n t i t a t i o n of the radioactivity indicated that the p r o p o r t i o n of APP770 m R N A was, o n the average, 1.3-3.2 times higher in A D t h a n in o t h e r n e u r o l o g i c a l disorders or control (Table IA). F u r t h e r more, the p r o p o r t i o n of APP751 m R N A was also higher,
TABLE II
APP mRNAs proportion in specific brain regions of carious disorders and fetus Disorder
A. PD
B. ALS
Age
Patient 1
67 years
Patient 2
72
Patient 1
62
Patient 2
70
Region
mRNA proportion (%)
Ratio (a + b)/c
APP770 (a)
APP751 (b)
APP695 (c)
Frontal cortex Midbrain Frontal cortex Midbrain
0.8 0.9 2.3 2.5
13.4 10.6 22.9 23.5
85.8 88.5 74.8 74.0
0.17 0.13 0.34 0.35
Frontal cortex Motor area Frontal cortex Motor area
4.2 3.7 3.9 4.0
27.6 24.8 20.9 24.2
68.2 71.5 75.2 71.8
0.47 0.40 0.33 0.39
C. SCD
57
Frontal cortex Cerebellum
1.1 0.6
22.8 11.2
76.1 88.2
0.31 0.13
D. Fetus
10 weeks
Whole brain
1.4
6.2
92.4
0.082
306
B
A
Mixed type
AD
4
123 367
"-
261
"-
C
1
D M ID
P D 1
2
1
2
2
Fr Mi Fr Mi
106 "93"-
49"-
F ALS
1
G
H
SCD
Infarction
Fr Ce
1 2
I
J
Hemorrhage
Control
Fetus
2
Fr Mo FrMo
1
2
1
2
3
4
5
Wh
367 261
1 0 6 m,93~
49~ Fig. 2. Autoradiograms of the RNase protection assay of APP mRNAs. The 367, 261 and 93 nt bands represe,, ,/0, APP7sl and APP695 mRNAs, respectively. The frontal cortex (Fr or no mark), midbrain (Mi), motor area (Mo), cerebellum (Ce) or whole brain (Wh) of patients with various neurological disorders, control persons, or fetus was examined. Representative cases are shown for each disorder group. A: AD (57, 69, 79 and 87 years of age). B: mixed-type dementia (74 and 84 years). C: MID (85 and 91 years). D: PD (67 and 72 years), E: ALS (62 and 70 years). F: SCD (57 years). G: cerebral infarction (74 and 85 years). H: cerebral hemorrhage (78 and 94 years). I: control (56, 76, 78, 82 and 97 years). J: fetus (10 weeks).
307 samples, as reported previously 41. These results indicated that the cerebral cortex Of AD was unique in the change in APP mRNAs proportion.
whereas that of APP695 mRNA was lower, in AD relative to other neurological disorders. Accordingly, the mean ratio of (APP770 m R N A + APP751 mRNA)/APP695 mRNA for AD exceeded that for other disorders. The difference in the ratio was statistically significant (P < 0.05) between AD and PD or control, while additional difference among other disorders did not reach a statistically significant level. We, then, compared the APP mRNAs proportion between the frontal cortex and other brain regions affected in each neurodegenerative disorder (Fig. 2DF, Table IIA-C). Neither autoradiography nor radioactivity quantitation revealed appreciable changes in the APP mRNAs proportion in the midbrain of PD. In the motor area of two ALS patients examined, the proportions of APP770 and APP751 mRNAs and thus the ratio of (APP770 mRNA + APP751 mRNA)/APP695 mRNA were as high as in AD patients. This result did not necessarily show specific change for ALS but rather exemplified the diversity among the individuals, because the proportion of APP770 and APP751 mRNAs in the frontal cortex, which apparently resembled the midbrain in the APP mRNAs expression (Table liB), was much lower in two other ALS patients (see Fig. 3). A nearly twice difference in the 367 nt band (APP770 mRNA) and the 261 nt band (APP751 mRNA) between the frontal cortex and the cerebellum of SCD was not specific for SCD, but was observed as well in other
Correlation between APP mRNAs proportion and age The effect of aging on APP mRNAs proportion is evident in autoradiograms of control samples arranged in the order of age (Fig. 21); the 261 nt band (APP751 mRNA) increased, whereas the 93 nt band (APP695 mRNA) decreased with the advance of age. The ratio (y) of (APP770 mRNA + APP751 mRNA)/APP695 mRNA showed a positive correlation with age (x) in both AD and non-AD groups (Fig. 3); y = 0.005x + 0.014 (r = 0.372) for the AD group and y = 0.004x0.037 (r = 0.486) for the non-AD group. The relationship between the ages of AD (XAo) and non-AD (Xnon-AD) giving the same ratio was XAD = 0 . 8 X n o n . A D -10.2, indicating that AD patients preceded non-AD persons by more than 20 years in terms of APP mRNA proportion. Referring to 90% confidence lines, we concluded that the ratio was considerably higher in the AD group than in the non-AD group throughout the age range examined. A comparison of samples.in seventies and eighties of age showed a statistically significant difference (P < 0.05) in the ratio between the two groups (Table IB). The APP mRNAs proportion changed also during development; the brain (as a whole) of 10-week fetus
0.60 o
E
AD group
0.50 "Jr
~o D. D.
/90% confidence areas are indicated with shadowing. The AD group includes AD and mixed-type dementia, o AD, (~) mixed-type dementia, • MID, • PD, • ALS, * SCD, • cerebral infarction, • cerebral hemorrhage, and * control.
308 showed the proportion much higher in APP695 mRNA and lower in APP751 mRNA than the adult brain (Fig. 2J, Table liD). DISCUSSION An important, though unknown, function of APP in the central nervous system has been hinted by several lines of evidence, such as neurotrophic 44'47 or neurotoxic activities of APP fragments 46'47. This idea led us to speculate that the change in APP mRNAs proportion al might be associated with neurodegenerative processes in general, irrespective of disorders. Our present study, however, argued against this possibility of universal degenerative process and indicated the change to be specific for AD. Our results revealed a correlation between the ratio of (APP770 mRNA + APP751 mRNA)/APP695 mRNA and age among elderly persons, both AD and non-AD, as reported by K6nig et al. preliminarily 17. The difference between AD and non-AD was temporal and not qualitative; the former reached the same level of the ratio more than 20 years earlier than the latter did. This finding is compatible with the well-known fact that the brain of persons without dementia exhibits changes similar to AD brain, such as senile plaques, neuronal cell loss and gliosis, to a lesser degree and at higher ages 43. In other words, our analysis proved, at a molecular level, that AD is a state in which the brain undergoes a series of age-associated physiological changes but prematurely. Thus, the ratio of KPI-haboring to lacking types of APP mRNAs may serve as a molecular index of aging of the brain. Changes in the proportion of APP mRNAs are presumably brought about by molecular as well as cellular mechanisms, viz., altered splicing of the APP gene transcript and altered composition of cell population. In support of the first mechanism, Johnson et al. 12 showed, by using the in situ hybridization technique, that an increase in the ratio of APP751 mRNA/APP695 mRNA in AD brain resulted from altered proportions of APP mRNAs in individual neurons but not from a decrease in APP695 mRNA-producing neurons. Lately, an increasing number of proteins of physiological importance have been shown to be generated by alternative splicing of a single gene transcript, and the splicing to be regulated developmentally and tissue-specifically 2. Alternative splicing of some gene transcripts is also known to change during physiological aging or under certain pathological conditions. For example, the splicing of fibronectin gene transcript changes in rat tissues during aging or in human cell lines in cellular senescence 2~'32, and that of myosin heavy-chain
gene transcript changes in smooth muscles with arteriosclerosis t9. Cytokines, such as transforming growth factor/31, and retinoic acid also alter the splicing of fibronectin gene transcript 21. It is of Interest that the latter agent induces not only cell differentiation but also an increase in the proportion of APP695 m R N A in SH-SY5Y neuroblastoma cells x6. Although neither mechanism nor factor responsible for the regulation of alternative splicing have been known, it is conceivable that a factor regulating the splicing of APP gene transcript undergoes prematurely an age-associated change in the AD brain. Recently, Oyama et al. 31 reported a correlation between expressions of APP751 mRNA and four-repeat type tau protein mRNA, indicating a common factor regulating the splicing of the two gene transcripts. The second mechanism is supported, in view of neuronal cell loss and reactive gliosis characteristic of AD brain, by the finding of cell-specific expression of APP gene transcript; APP695 mRNA prevailed in cultured neuronal cells, whereas APP770 and APP751 mRNAs predominated in astrocyte- or microglia-enriched cultures 29. A similar gerontological change in the composition of cell types 43 may also explain the age-related change in the APP mRNAs proportion. Our observation that the proportion remained much the same even in affected brain regions in various neurodegenerative disorders does not necessarily exclude this mechanism, because the degree of change in cell population differs considerably between AD and other disorders. Although several researchers have reported aberrant proportions of APP mRNAs in AD brain, the results are controversial. The reports of an increase in the APP751/APP695 mRNA ratio 1°-12 or a selective reduction of APP695 mRNA with no concomitant change in APP770 or APPvs I mRNA in AD brain 27'3a are essentially consistent with our results 4°'41. On the other hand, the reports showing no selective alteration of APP m R N A expression in the cerebral cortex of AD 17'18'31"33 or an increase, rather than a decrease, of APP695 mRNA in some specific brain regions 7"33 are contradictory. The discrepancy among these reports may be due to the difference in methodology employed a n d / o r samples analyzed; our earlier discussion is indicative of a considerable variability of APP mRNAs proportion with the age in both AD and control groups. Furthermore, AD has been suggested as an etiologically heterogeneous disorder caused by various mechanisms of amyloid deposition 39. It is noteworthy that transgenic mice transfected with APP751 cDNA developed amyloid deposition, while APP69s cDNA-transfected mice did not 3s.
309 Recently, mRNA encoding APRP563 (amyloid precursor-related protein), an APP751 variant, was discovered4, and its possible contribution to the increase in APP75x mRNA in AD brain was suggested 28. Our previous analysis4° using the Northern blotting technique failed to detect APRP563 mRNA, indicating that its content was, if any, very little. Because the probe used in the present study was unable to distinguish between APRP56 ~ mRNA and APP75~ mRNA, furthei" analysis with APRPs63-specific probe is necessary for precise estimation Of the significanCeof this variarit, i Information about sites and enzymes responsible for physiological APP cleavage is of crucial importance to an understanding of amyloidogenic mechanism. Ishiura et ai. s'9 proposed two serine proteases, multicatalytic proteinase (ingensin) and prolyl endopeptidase, as candidates of the enzymes incising APPs at amino- and carboxyl-termini, respectively, of/3A4 protein. On the other hand, Anderson et al. l showed that APP degradation in neuronal PC-12 cells started in the interior, between Lys t6 and Leu iT, of flA4 protein. This finding, which confirmed previous reports concerning the initial cleavage sites of A P P s 5'36, is suggestive of the action of a protease with trypsin-like specificity. Interestingly, the KPI segment of APP exhibited particularly strong inhibition against trypsin and chymotrypsin15. In addition, the candidate for APP secretase showed trypsinlike activity 37. All these observations suggest possible involvement of serine proteases in APP degradation and their control by KPIs in the human brain. Most probably, an increased expression of KPI-harboring APPs perturbs the balance between the proteases and the inhibitors, and eventually leads to accumulation of an aberrant intermediate,/3A4 protein. Acknowledgements. This work was supported by Grants-in-Aid and a Special Grant for Clinical Investigation from the Ministry of Education, Science and Culture, Japan, and a grant from the Sasagawa Health Science Foundation. We thank Drs. M. Kameyama (Sumitomo Hospital), T. Takeda (Kyoto University) and K. Uragami (Tottori University) for useful discussion, and Dr. B.R. Das (Institute of Life Sciences, India) for collaborative work. Drs. M. Ogawa, F. Udaka, K. Hara and R. Matsumoto (Kyoto University-affiliated hospitals) are gratefully acknowledged for providing us with brain samples.
REFERENCES 1 Anderson, J.P., Esch, F.S., Keim, P.S., Sambamurti, K., Lieberburg, I. and Robakis, N.K., Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 Cells, Neurosci. Lett., 128 (1991) 126-128. 2 Breitbart, R.E., Andreadis, A. and NadaI-Ginard, B., Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes, Annu. Rev. Biochem., 56 (1987) 467-495. 3 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299.
4 De Sauvage, F. and Octave, J.-N., A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein, Science, 245 (1989) 651-653, 5 Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J., Cleavage of amyloid fl peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. 6 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 7 Golde, T.E., Estus, S., Usiak, M., Younkin, L.H. and Younkin, S.G., Expression of fl amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR, Neuron, 4 (1990) 253-267. 8 Ishiura, S., Tsukahara, T., Tabira; T. and Sugita, H., Putative N-terminal splitting enzyme of amyloid A4 peptides is the ~multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells, FEBS Lett., 257 (1989) 388-392. 9 Ishiura, S., Tsukahara, T., Tabira, T., Shimizu, T.I~Arahata, K: and Sugita, H., Identification of a putative amyloid A4-generating enzyme as a prolyl endopeptidase, FEBS Left., 260 (1990) 131134. 10 Johnson, S.A., Pasinetti, G.M., May, P.C., Ponte, P.A., Cordell, B. and Finch, C.E., Selective reduction of mRNA for the flamyloid precursor protein that lacks a Kunitz-type protease inhibitor motif in cortex from AIzheimer brains, Exp. Neurol., 102 (1988) 264-268. 11 Johnson, S.A., Rogers, J. and Finch, C.E., APP-695 transcript prevalence is selectively reduced during Alzheimer's disease in cortex and hippocampus but not in cerebellum, Neurobiol. Aging, 10 (1989) 267-272. 12 Johnson, S.A., McNeill, T., Cordell, B. and Finch, C.E., Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer's disease, Science, 248 (1990) 854-857. 13 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and MiillerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. 14 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530-532. 15 Kitaguchi, N., Takahashi, Y., Oishi, K., Shiojiri, S, Tokushima, Y., Utsunomiya, T. and Ito, H., Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of AIzheimer's disease: different properties compared with protease nexin I, Biochim. Biophys. Acta, 1038 (1990) 105-113. 16 K6nig, G., Masters, C.L. and Beyreuther, K., Retinoic acid induced differentiated neuroblastoma cells show increased expression of the A4 amyloid gene of Alzheimer's disease and an altered splicing pattern, FEBS Lett., 269 (1990) 305-310. 17 K6nig, G., Salbaum, J.M., Wiestler, O., Lang, W., Schmitt, H.P., Masters, C.L. and Beyreuther, K., Alternative splicing of the flA4 amyloid gene of Alzheimer's disease in cortex of control and Alzheimer's disease patients, Mol. Brain Res., 9 (1991) 259-262. 18 Koo, E.H., Sisodia, S.S., Cork, L.C., Unterbeck, A., Bayney, R.M. and Price, D.L., Differential expression of amyloid precursor protein mRNAs in cases of Alzheimer's disease and in aged nonhuman primates, Neuron, 2 (1990) 97-104. 19 Kuro-o, M., Nagai, R., Nakahara, K., Katoh, H., Tsai, R.-C., Tsuchimochi, H., Yazaki, Y., Ohkubo, A. and Takaku, F., cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis, J. Biol. Chem., 266 (1991) 3768-3773. 20 Lemaire, H.G., Salbaum, J.M., Multhaup, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K. and Miiller-Hill, B., The preA4695 precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons, Nucl. Acids Res., 17 (1989) 517-522. 21 Magnuson, V.L., Young, M., Schattenberg, D.G., Mancini, M.A., Chen, D., Steffensen, B. and Klebe, R.J., The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors, J. Biol. Chem., 266 (1991) 14654-14662.
310 22 Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.N. and Beyreuther, K., Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels, EMBO Z, 4 (1985) 2757-2763. 23 Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K., Amyloid plaque core protein in Alzheimer disease and Down syndrome, Proc. Natl. Acad. Sci. USA, 82 (1985) 4245-4249. 24 McKahnn, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease, Neurology, 34 (1984) 939-944. 25 Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor, Nucl. Acids Res., 12 (1984) 7035-7058. 26 Mountjoy, C.Q., Tomlinson, B.E. and Gibson, P.H., Amyloid and senile plaques and cerebral blood vessels. A semi-quantitative investigation of a possible relationship, 3~ Neurol. Sci., 57 (1982) 89-103. 27 Neve, R.L., Finch, E.A. and Dawes, L.R., Expression of the Alzheimer amyloid precursor gene transcripts in the human brain, Neuron, 1 (1988) 669-677. 28 Neve, R.L., Rogers, J. and Higgins, G.A., The Alzheimer amyloid precursor-related transcript lacking the/3/A4 sequence is specifically increased in Alzheimer's disease brain, Neuron, 5 (1990) 329-338. 29 Ohyanagi, Y., Takahashi, K., Kamegai, M. and Tabira, T., Developmental and differential expression of beta amyloid protein precursor mRNAs in mouse brain, Biochem. Biophys. Res. Commun., 167 (1990) 54-60. 30 Oppenheimer, D.R., Disease of the basal ganglia, cerebellum and motor neurons. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen (Eds.), Gleenfield's Neuropathology, 4th edn., Edward Arnold, London, 1984, pp. 699-747. 31 Oyama, F., Shimada, H., Oyama, R., Titani, K. and Ihara, Y., Differential expression of/3 amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP-751 and four-repeat tau, J. Neuropathol. Exp. Neurol., 50 (1991) 560-578. 32 Pagani, F., Zagato, L., Vergani, C., Casari, G., Sidoli, A. and Baralle, F.E., Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat, J. Cell Biol., 113 (1991) 1223-1229. 33 Palmert, M.R., Golde, T.E., Cohen, M.L., Kovacs, D.M., Tanzi, R.E., Gusella, J.F., Usiak, M.F., Younkin, L.H. and Younkin, S.G., Amyloid protein precursor messenger RNAs: differential expression in Alzheimer's disease, Science, 241 (1988) 1080-1084. 34 Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F. and Cordell, B., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature, 331 (1988) 525-527. 35 Quon, D., Wang, Y., Catalano, R., Scardina, J.M., Murakami. K. and Cordell, B., Formation of /3-amyloid protein deposits in brains of transgenic mice, Nature, 352 (1991) 239-241.
36 Sisodia, S.S., Koo, E.H., Beyreuther, K., Unterbeck, A. and Price, D.L., Evidence that /3-amyloid protein in Alzheimer's disease is not derived by normal processing, Science, 248 (1990) 492-495. 37 Small, D.H., Moir, R.D., Fuller, S.J., Michaelson, S., Bush, A.I., Li, Q.-X., Milward, E., Hilbich, C., Weidemann, A., Beyreuther, K. and Masters, C.L., A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of Alzheimer's disease, Biochemistry, 30 (1991) 10795-10799. 38 Spillantini, M.G., Hunt, S.P., Ulrich, J. and Goedert, M., Expression and cellular localization of amyloid /3-protein precursor transcripts in normal human brain and in Alzheimer's disease, Mol. Brain Res., 6 (1989) 143-150. 39 St. George-Hyslop, P.H., Haines, J.L., Farrer, L.A., Polinsky, R., Van Broeckhoven, C., Goate, A., McLachlan, D.R.C., Orr, H., Bruni, A.C., Sorbi, S., Rainero, 1., Foncin, J.-F., Pollen, D., Cantu, J-M., Tupler, R., Voskresenskaya, N., Mayeux, R., Growdon, J., Fried, V.A., Myers, R.H., Nee, L., Backhovens, H., Martin, J-J., Rossor, M., Owen, M.J., Mullan, M., Percy, M.E., Karlinsky, H., Rich, S., Heston, L., Montesi, M., Mortilla, M., Nacmias, N., Gusella, J.F., Hardy, J.A. and other members of the FAD Collaborative Study Group, Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder, Nature, 347 (1990) 194-197. 40 Tanaka, S., Nakamura, S., Ueda, K., Kameyama, M., Shiojiri, S., Takahashi, Y., Kitaguchi, N. and Ito, H., Three types of amyloid protein precursor mRNA in human brain: their differential expression in Alzheimer's disease, Biochem. Biophys. Res. Commun., 157 (1988) 472-479. 41 Tanaka, S., Shiojiri, S., Takahashi, Y., Kitaguchi, N., lto, H., Kameyama, M., Kimura, J., Nakamura, S. and Ueda, K., Tissuespecific expression of three types of/3-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain, Biochern. Biophys. Res. Commun., 165 (1989) 14061414. 42 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J.F. and Neve, R.L., Protease inhibitor domain encoded by an amytoid protein precursor mRNA associated with Alzheimer's disease, Nature, 331 (1988) 528-530. 43 Tomlinson, B.E. and Corsellis, J.A.N., Ageing and the dementias. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen (Eds.), Gleenfield's Neuropathology, 4th edn., Edward Arnold, London, 1984, pp. 951-1025. 44 Whitson, J.S., Selkoe, D.J. and Cotman, C.W., Amyloid /3 protein enhances the survival of hippocampal neurons in vitro, Science, 243 (1989) 1488-1490. 45 Wong, C.W., Quaranta, V. and Glenner, G.G., Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related, Proc. Natl. Acad. Sci. USA, 82 (1985) 8729-8732. 46 Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L. and Neve, R.L., Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease, Science, 245 (1989) 417-420. 47 Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotrophic and neurotoxic effects of amyloid/3 protein: reversal by tachykinin neuropeptides, Science, 250 (1990) 279-282. 48 Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H. and Sakaki, Y., Genomic organization of the human amyloid beta-protein precursor gene, Gene, 87 (1990) 257-263.
303
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00 BRESM 70486
Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimer's disease and other neurological disorders Seigo Tanaka
a
Li Liu
b
Jun Kimura
b
S a t o s h i S h i o j i r i c, Y a s u y u k i T a k a h a s h i
N o b u y a K i t a g u c h i c, S h i g e n o b u N a k a m u r a
d and Kunihiro Ueda
c,
a
a Department of Clinical Science and Laboratory Medicine, b Department of Neurology, Kyoto University Faculty of Medicine, Kyoto (Japan), c Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry, Shizuoka (Japan) and d 3rd Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan)
(Accepted 19 May 1992)
Key words: Alzheimer'sdisease;/3A4 amyloid;Amyloidprecursor protein; Alternative splicing; Protease inhibitor; Aging;
RNase protection assay
In the human brain, alternative splicing of amyloid precursor protein (APP) gene transcript generates at least three types of mRNA coding for APP770, APP751 and APP695.The former two types harbor, but the latter'one lacks a domain of Kunitz-type serine protease inhibitor (KPI). We studied, by using the RNase protection technique, the expression of APP mRNAs in brains of Alzheimer's disease (AD) and other neurological disorders with special reference to aging. We found that the ratio of (APP770 mRNA+APP751 mRNA)/APP695 mRNA in the frontal cortex increased approximately 1.5-fold in AD compared with other neurodegenerative or cerebrovascular disorders. The ratio in other neurological disorders did not change significantly from control even in their affected brain regions. On the other hand, we found a positive correlation between the ratio and age; the ratio (y) increased gradually with the advance of age (x) as expressed by y = 0.005x +0.014 (r = 0.372) for the AD group, and y = 0.004x -0.037 (r = 0.486) for the non-AD group. These correlations indicate that the AD brain reached the same ratio of KPl-harboring to lacking APP mRNAs a few decades earlier than the non-AD brain in senescence. This finding of AD-specificand age-related change led us to the idea that a relative increase in KPI-harboringAPPs over a KPI-lacking APP may perturb normal degradation of APPs, thereby leading to deposition of/3A4 protein as amyloid.
INTRODUCTION Alzheimer's disease (AD) constitutes one of the most common neurodegenerative disorders, presenting as progressive dementia with loss of memory, intellectual disturbance, and language problems. The pathogenesis of this disease, although unknown, evolves around the deposition o f / 3 A 4 protein in senile plaque cores and cerebral vessels 6'22'23'26'45.The flA4 protein is generated from larger precursors (amyloid precursor proteins; APPs) that have structual features of cell surface receptors ~3. Three (or reportedly four) types of APP m R N A [APP770, APP751, (APPTt 4) and APP695 mRNAs] are produced from a single gene transcript by alternative splicing of exon 7 and 8 segments 2°'4s. The 56 amino acid sequence encoded by exon 7 is highly homologous to a Kunitz-type serine protease inhibitor
(KPI)14'34'42 and
has a protease inhibitor activity, as demonstrated by an in vitro expression experiment 14. This KPI segment is harbored in APP770 and APP751, but not in APP695 (nor APP714). Our previous study 41 showed that the proportion of APP770 m R N A (or APP770 m R N A + APP751 m R N A ) in various brain regions is higher in A D than in control. The highest level was observed in the cerebral cortex and hippocampus known to have the highest density of senile plaques. In addition, A D samples showing histologically a high density of senile plaques exhibited a high ratio of (APP770 m R N A + APP751 m R N A ) / A P P 6 9 5 m R N A 1~'41. These results led us to an inference that an increased expression of KPI-harboring APPs relative to KPI-lacking one(s) might affect normal degradation of APPs in the brain and lead to accumulation of aberrant proteolytic products as amyloid.
Correspondence: S. Tanaka, Department of Clinical Science and Laboratory Medicine, KyotoUniversity Faculty of Medicine, Shogoin, Sakyo-ku,
Kyoto 606, Japan. Fax: (81) 75-771-4792.
304 Neurodegenerative changes are observed in Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and spinocerebellar degeneration (SCD), in addition to AD. These diseases share, more or less, common pathological changes such as a gradual decay and loss of neurons accompanied by gliosis at specific brain regions 3°. Senile plaques increase with the advance of age in patients with these disorders as well as persons with no neurological disorder, although to a much lesser degree than in AD patients. In view of a good correlation between the density of senile plaques and APP mRNAs proportion ~2'4t, we addressed the following questions in the present study: (1) Do A P P 7 7 0 and APP751 mRNAs increase in other neurological disorders? (2) Does the APP mRNAs proportion change with aging? And (3) does the APP mRNAs proportion differ between the AD group and the non-AD group in an age-matched comparison? Our results indicate that AD exhibits a unique change in the APP mRNAs expression, not shared by other neurological disorders until far later senescence, in the brain. MATERIALS A N D METHODS Brain samples and total RNA isolation Postmortem brain samples were obtained, within 12 h after death, from patients with AD, mixed-type dementia, multi-infarct dementia (MID), PD, ALS, SCD ( O P C A type), cerebral infarction and cerebral hemorrhage, and persons without neurological disorders (control) (Table IA). O n e half of the brain, frozen at - 8 0 ° C , was used for m R N A analysis, and the other half, fixed in formalin, for histological examination. We diagnosed A D clinically using the N I N C D S - A D R D A criteria 24 and histologically by the density of
senile plaques and neurofibrillary tangles stained with the silver impregnation method. MID patients showed histologically multiple cerebral infarction with atherosclerosis and arteriolar sclerosis as underlying vascular lesions. Mixed-type dementia is a state exhibiting both AD-type changes and vascular lesions. We made diagnosis of other disorders clinically and confirmed the absence of AD-type changes by histological examination. The midbrain of PD patients showed loss of substantia nigral cells and gliosis with Lewy body formation. The motor area (Brodmann area 4) of two patients with ALS showed mild loss of giant pyramidal cells of Betz. The cerebellar cortex of SCD patient showed loss of Purkinje cells. We cut out the frontal cortex (Brodmann areas 9 and 10) from the frozen brain of all patients, and also specific brain regions affected in each neurodegenerative disorder mentioned above. Sample of patients with cerebral infarction or hemorrhage was from the region devoid of major lesions. We obtained sample also from the whole brain of fetus aborted at 10 weeks. Total cellular R N A was extracted from brain samples by the g u a n i d i n i u m / C s C I method 3.
RNase protection assay The antisense R N A probe for RNase protection assay 25 (Fig. 1) was prepared as follows. A Ddel (nt 7 7 4 ) - X h o i (nt 1136)fragment of APP770 c D N A 14 encompassing exons 7 and 8 was inserted inversely into pSP64 plasmid (Promega) downstream to the SP6 promotor site, and the plasmid was proliferated in Escherichia coli. Using the EcoR1 fragment of the plasmid D N A (1 /xg) as template, antisense R N A fragment was synthesized at 40°C for 30 min with 5 units of SP6 R N A polymerase (BRL) in 10 txl of the reaction mixture [40 m M Tris-Cl (pH 7.5), 6 m M MgCI2, 2 m M spermidine, 0.5 m M each ATP, U T P and GTP, and 12.5 ~ M [a- 32p]CTP (400 Ci/mmol)]. The mixture was then supplemented with 1 /~g of RNase-free DNase I (BRL), and further incubated at 37°C for 10 rain. R N A was extracted from the mixture with phenol/chloroform, and precipitated with ethanol. The R N A probe thus 32p-labelled (5 × 105 cpm) was hybridized with sample R N A (10 ~tg) in 3 0 / z l of the hybridization buffer [80% formamide, 40 m M PIPES (pH 6.4), 400 m M NaCI, and 1 m M EDTA] at 45°C for 12 h. The hybridization mixture was treated at 25°C for 10 min with RNase A (Sigma; 40 t z g / m l ) and RNase T1 (BRL; 2 ~zg/ml) in 300 tzl of the digestion buffer [10 m M Tris-Cl (pH 7.5), 300 m M NaCI, and 5 m M EDTA]. After addition of 20 tzl of 10% SDS and 10 #1 of proteinase K (Merck; 5 m g / m l ) to
TABLE 1
APP mRNAs proportion in the frontal cortex of t:arious disorders * Proportions were calculated from radioactivities of band areas located by autoradiography (Fig. 2) as described in Materials and Methods. ** Samples obtained from persons in seventies and eighties of age were compared. The A D group includes A D and mixed-type dementia. Values are mean -+ S.D.
Disorder or group
n
Age (years)
mRNA proportion * (%)
Ratio
APP77o
APP751
App~o5
~a)
(b)
(c)
3.5_+ 1.9 2.2 _+0.5 2.4 _+ 1.6 1.9-+ 1.0 2.6 _+ 1.7 1.1 1.7 _+0.8 a 1.4 _+0.8 1.7 + 0.7 a
25.2_+5.3 23.2 _+4.7 22.4 _+4.9 18.8-+4.8 19.4 _+6.2 22.8 20.6 _+6.4 20.0 _+6.1 19.0 + 5.6
71.3_+6.9 74.7 _+4.2 75.2 _+5.8 79.3_+5.7 78.0 _+7.8 76.1 77.7 _+6.0 78.5 _+5.9 79.3 + 6.2 "
0.41 _+0.12 0.34 _+0.07 0.34 ± 0.11 0.27_+0.09 ~' 0.30 5:0.13 0.31 0.29 5:0.09 0.28 5:0.09 0.27 ± 0.11 a
B. Age-matched comparison between A D and non-AD groups ** A D group 8 78.9_+ 5.2 3.2_+2.0 N o n - A D group 21 77.9_+ 5.1 1.7_+ 1.1 b
24.3_+5.4 20.6_+4.8
72.4_+7.(I 77.7_+5.1 b
0.39_+0.13 0.29_+0.08 b
A. Comparison among neurological disorders AD 8 74.8_+ 9.2 Mixed-type dementia 2 79.0 _+ 7.1 MID 6 81.5 _+ 11.1 PD 5 70.6-+ 2.7 ALS 4 65.0 _+ 7.4 SCD 1 57.0 Infarction 6 78.2 -+ 4.7 Hemorrhage 4 85.5 -+ 6.6 Control 7 75.3 -+ 13.5
P < 0.05 (Student's t-test), compared with AD; b p < 0.05, compared with A D group.
(a + b) / c
305 regression method to analyze correlation between the ratio and age. The results were expressed as a regression line accompanied by 90% confidence lines and a correlation coefficient (r).
KPI segment !
APP gene transcript
RESULTS
APP,,0
l/ I 0
Proportion o f A P P m R N A s in the brain
367nt
T h e R N a s e p r o t e c t i o n assay of b r a i n A P P m R N A s revealed four b a n d s in a u t o r a d i o g r a p h y at the position
261nt APPT14 mRNA
/
~
m R N A s e n c o d i n g APP770, APP751, APP69s, a n d APP7s 1 plus APP695, respectively (Fig. 1); the 93 n t b a n d was
/ 93 nt
APP695 mRNA
of 367, 261, 93 a n d 49 nt long (Fig. 2), c o r r e s p o n d i n g to
49nt
/~r'~-]~_ 93nt
i n t e r p r e t e d to r e p r e s e n t solely APP69 s m R N A , b e c a u s e
106 nt
/ 49nt
Fig. 1. Structures of APP gene transcript and four types of spliced APP mRNAs in relation to the sites hybridizing with antisense RNA probe in the RNase protection assay. The number of each domain corresponds to that of exon in the APP gene. Exon 7 encodes the KPI segment. Solid bars accompanied by lengths in nucleotides (nt) represent the fragments of the probe protected from RNase digestion.
the solution, the mixture was incubated at 37°C for 15 min. RNA was extracted with phenol/chloroform and precipitated with ethanol, and the pellet was dissolved in the loading buffer [80% formamide, 1 mM EDTA (pH 8.0), 0.1% bromophenol blue, and 0.1% xylene cyanol]. RNA hybrids were denatured, and electrophoresed on 6% polyacrylamide/10 M urea gel. Finally, we subjected the gel to autoradiography using an intensifying screen and Kodak X-Omat film. For quantitative analysis, band areas located by autoradiography were cut out from dried gel, and their radioactivities were determined by the liquid scintillation method. The values thus obtained were corrected for the number of cytosine residues contained in each fragment for calculation of APP mRNAs proportion.
Statistical analysis We used Student's t-test to evaluate significance of a difference in the ratio of APP mRNAs between two groups, and the linear
a 106 n t b a n d expected for APP714 m R N A was u n d e tectable u n d e r o u r e x p e r i m e n t a l conditions. A m o n g the four b a n d s , the 367 nt b a n d was the least promin e n t a n d hardly d e t e c t a b l e in some samples unless a l o n g e r exposure of the gel. O t h e r m i n o r b a n d s emerging variably r e m a i n to be characterized. Q u a n t i t a t i v e analysis of radioactivities revealed that APP695 m R N A a b o u n d e d in the brain, followed by APP75 t m R N A (Table IA). APP770 m R N A was a m i n o r c o m p o n e n t , regardless of b r a i n regions.
Difference in the A P P m R N A s proportion a m o n g neurological disorders W e investigated the difference in the A P P m R N A s p r o p o r t i o n a m o n g various n e u r o l o g i c a l disorders, first, in an identical b r a i n region, i.e., the frontal cortex (Fig. 2 A - I ) . As a distinct finding, the a u t o r a d i o g r a m s of several A D samples exhibited a d e n s e 367 nt b a n d (APP770 m R N A ) . Q u a n t i t a t i o n of the radioactivity indicated that the p r o p o r t i o n of APP770 m R N A was, o n the average, 1.3-3.2 times higher in A D t h a n in o t h e r n e u r o l o g i c a l disorders or control (Table IA). F u r t h e r more, the p r o p o r t i o n of APP751 m R N A was also higher,
TABLE II
APP mRNAs proportion in specific brain regions of carious disorders and fetus Disorder
A. PD
B. ALS
Age
Patient 1
67 years
Patient 2
72
Patient 1
62
Patient 2
70
Region
mRNA proportion (%)
Ratio (a + b)/c
APP770 (a)
APP751 (b)
APP695 (c)
Frontal cortex Midbrain Frontal cortex Midbrain
0.8 0.9 2.3 2.5
13.4 10.6 22.9 23.5
85.8 88.5 74.8 74.0
0.17 0.13 0.34 0.35
Frontal cortex Motor area Frontal cortex Motor area
4.2 3.7 3.9 4.0
27.6 24.8 20.9 24.2
68.2 71.5 75.2 71.8
0.47 0.40 0.33 0.39
C. SCD
57
Frontal cortex Cerebellum
1.1 0.6
22.8 11.2
76.1 88.2
0.31 0.13
D. Fetus
10 weeks
Whole brain
1.4
6.2
92.4
0.082
306
B
A
Mixed type
AD
4
123 367
"-
261
"-
C
1
D M ID
P D 1
2
1
2
2
Fr Mi Fr Mi
106 "93"-
49"-
F ALS
1
G
H
SCD
Infarction
Fr Ce
1 2
I
J
Hemorrhage
Control
Fetus
2
Fr Mo FrMo
1
2
1
2
3
4
5
Wh
367 261
1 0 6 m,93~
49~ Fig. 2. Autoradiograms of the RNase protection assay of APP mRNAs. The 367, 261 and 93 nt bands represe,, ,/0, APP7sl and APP695 mRNAs, respectively. The frontal cortex (Fr or no mark), midbrain (Mi), motor area (Mo), cerebellum (Ce) or whole brain (Wh) of patients with various neurological disorders, control persons, or fetus was examined. Representative cases are shown for each disorder group. A: AD (57, 69, 79 and 87 years of age). B: mixed-type dementia (74 and 84 years). C: MID (85 and 91 years). D: PD (67 and 72 years), E: ALS (62 and 70 years). F: SCD (57 years). G: cerebral infarction (74 and 85 years). H: cerebral hemorrhage (78 and 94 years). I: control (56, 76, 78, 82 and 97 years). J: fetus (10 weeks).
307 samples, as reported previously 41. These results indicated that the cerebral cortex Of AD was unique in the change in APP mRNAs proportion.
whereas that of APP695 mRNA was lower, in AD relative to other neurological disorders. Accordingly, the mean ratio of (APP770 m R N A + APP751 mRNA)/APP695 mRNA for AD exceeded that for other disorders. The difference in the ratio was statistically significant (P < 0.05) between AD and PD or control, while additional difference among other disorders did not reach a statistically significant level. We, then, compared the APP mRNAs proportion between the frontal cortex and other brain regions affected in each neurodegenerative disorder (Fig. 2DF, Table IIA-C). Neither autoradiography nor radioactivity quantitation revealed appreciable changes in the APP mRNAs proportion in the midbrain of PD. In the motor area of two ALS patients examined, the proportions of APP770 and APP751 mRNAs and thus the ratio of (APP770 mRNA + APP751 mRNA)/APP695 mRNA were as high as in AD patients. This result did not necessarily show specific change for ALS but rather exemplified the diversity among the individuals, because the proportion of APP770 and APP751 mRNAs in the frontal cortex, which apparently resembled the midbrain in the APP mRNAs expression (Table liB), was much lower in two other ALS patients (see Fig. 3). A nearly twice difference in the 367 nt band (APP770 mRNA) and the 261 nt band (APP751 mRNA) between the frontal cortex and the cerebellum of SCD was not specific for SCD, but was observed as well in other
Correlation between APP mRNAs proportion and age The effect of aging on APP mRNAs proportion is evident in autoradiograms of control samples arranged in the order of age (Fig. 21); the 261 nt band (APP751 mRNA) increased, whereas the 93 nt band (APP695 mRNA) decreased with the advance of age. The ratio (y) of (APP770 mRNA + APP751 mRNA)/APP695 mRNA showed a positive correlation with age (x) in both AD and non-AD groups (Fig. 3); y = 0.005x + 0.014 (r = 0.372) for the AD group and y = 0.004x0.037 (r = 0.486) for the non-AD group. The relationship between the ages of AD (XAo) and non-AD (Xnon-AD) giving the same ratio was XAD = 0 . 8 X n o n . A D -10.2, indicating that AD patients preceded non-AD persons by more than 20 years in terms of APP mRNA proportion. Referring to 90% confidence lines, we concluded that the ratio was considerably higher in the AD group than in the non-AD group throughout the age range examined. A comparison of samples.in seventies and eighties of age showed a statistically significant difference (P < 0.05) in the ratio between the two groups (Table IB). The APP mRNAs proportion changed also during development; the brain (as a whole) of 10-week fetus
0.60 o
E
AD group
0.50 "Jr
~o D. D.
/90% confidence areas are indicated with shadowing. The AD group includes AD and mixed-type dementia, o AD, (~) mixed-type dementia, • MID, • PD, • ALS, * SCD, • cerebral infarction, • cerebral hemorrhage, and * control.
308 showed the proportion much higher in APP695 mRNA and lower in APP751 mRNA than the adult brain (Fig. 2J, Table liD). DISCUSSION An important, though unknown, function of APP in the central nervous system has been hinted by several lines of evidence, such as neurotrophic 44'47 or neurotoxic activities of APP fragments 46'47. This idea led us to speculate that the change in APP mRNAs proportion al might be associated with neurodegenerative processes in general, irrespective of disorders. Our present study, however, argued against this possibility of universal degenerative process and indicated the change to be specific for AD. Our results revealed a correlation between the ratio of (APP770 mRNA + APP751 mRNA)/APP695 mRNA and age among elderly persons, both AD and non-AD, as reported by K6nig et al. preliminarily 17. The difference between AD and non-AD was temporal and not qualitative; the former reached the same level of the ratio more than 20 years earlier than the latter did. This finding is compatible with the well-known fact that the brain of persons without dementia exhibits changes similar to AD brain, such as senile plaques, neuronal cell loss and gliosis, to a lesser degree and at higher ages 43. In other words, our analysis proved, at a molecular level, that AD is a state in which the brain undergoes a series of age-associated physiological changes but prematurely. Thus, the ratio of KPI-haboring to lacking types of APP mRNAs may serve as a molecular index of aging of the brain. Changes in the proportion of APP mRNAs are presumably brought about by molecular as well as cellular mechanisms, viz., altered splicing of the APP gene transcript and altered composition of cell population. In support of the first mechanism, Johnson et al. 12 showed, by using the in situ hybridization technique, that an increase in the ratio of APP751 mRNA/APP695 mRNA in AD brain resulted from altered proportions of APP mRNAs in individual neurons but not from a decrease in APP695 mRNA-producing neurons. Lately, an increasing number of proteins of physiological importance have been shown to be generated by alternative splicing of a single gene transcript, and the splicing to be regulated developmentally and tissue-specifically 2. Alternative splicing of some gene transcripts is also known to change during physiological aging or under certain pathological conditions. For example, the splicing of fibronectin gene transcript changes in rat tissues during aging or in human cell lines in cellular senescence 2~'32, and that of myosin heavy-chain
gene transcript changes in smooth muscles with arteriosclerosis t9. Cytokines, such as transforming growth factor/31, and retinoic acid also alter the splicing of fibronectin gene transcript 21. It is of Interest that the latter agent induces not only cell differentiation but also an increase in the proportion of APP695 m R N A in SH-SY5Y neuroblastoma cells x6. Although neither mechanism nor factor responsible for the regulation of alternative splicing have been known, it is conceivable that a factor regulating the splicing of APP gene transcript undergoes prematurely an age-associated change in the AD brain. Recently, Oyama et al. 31 reported a correlation between expressions of APP751 mRNA and four-repeat type tau protein mRNA, indicating a common factor regulating the splicing of the two gene transcripts. The second mechanism is supported, in view of neuronal cell loss and reactive gliosis characteristic of AD brain, by the finding of cell-specific expression of APP gene transcript; APP695 mRNA prevailed in cultured neuronal cells, whereas APP770 and APP751 mRNAs predominated in astrocyte- or microglia-enriched cultures 29. A similar gerontological change in the composition of cell types 43 may also explain the age-related change in the APP mRNAs proportion. Our observation that the proportion remained much the same even in affected brain regions in various neurodegenerative disorders does not necessarily exclude this mechanism, because the degree of change in cell population differs considerably between AD and other disorders. Although several researchers have reported aberrant proportions of APP mRNAs in AD brain, the results are controversial. The reports of an increase in the APP751/APP695 mRNA ratio 1°-12 or a selective reduction of APP695 mRNA with no concomitant change in APP770 or APPvs I mRNA in AD brain 27'3a are essentially consistent with our results 4°'41. On the other hand, the reports showing no selective alteration of APP m R N A expression in the cerebral cortex of AD 17'18'31"33 or an increase, rather than a decrease, of APP695 mRNA in some specific brain regions 7"33 are contradictory. The discrepancy among these reports may be due to the difference in methodology employed a n d / o r samples analyzed; our earlier discussion is indicative of a considerable variability of APP mRNAs proportion with the age in both AD and control groups. Furthermore, AD has been suggested as an etiologically heterogeneous disorder caused by various mechanisms of amyloid deposition 39. It is noteworthy that transgenic mice transfected with APP751 cDNA developed amyloid deposition, while APP69s cDNA-transfected mice did not 3s.
309 Recently, mRNA encoding APRP563 (amyloid precursor-related protein), an APP751 variant, was discovered4, and its possible contribution to the increase in APP75x mRNA in AD brain was suggested 28. Our previous analysis4° using the Northern blotting technique failed to detect APRP563 mRNA, indicating that its content was, if any, very little. Because the probe used in the present study was unable to distinguish between APRP56 ~ mRNA and APP75~ mRNA, furthei" analysis with APRPs63-specific probe is necessary for precise estimation Of the significanCeof this variarit, i Information about sites and enzymes responsible for physiological APP cleavage is of crucial importance to an understanding of amyloidogenic mechanism. Ishiura et ai. s'9 proposed two serine proteases, multicatalytic proteinase (ingensin) and prolyl endopeptidase, as candidates of the enzymes incising APPs at amino- and carboxyl-termini, respectively, of/3A4 protein. On the other hand, Anderson et al. l showed that APP degradation in neuronal PC-12 cells started in the interior, between Lys t6 and Leu iT, of flA4 protein. This finding, which confirmed previous reports concerning the initial cleavage sites of A P P s 5'36, is suggestive of the action of a protease with trypsin-like specificity. Interestingly, the KPI segment of APP exhibited particularly strong inhibition against trypsin and chymotrypsin15. In addition, the candidate for APP secretase showed trypsinlike activity 37. All these observations suggest possible involvement of serine proteases in APP degradation and their control by KPIs in the human brain. Most probably, an increased expression of KPI-harboring APPs perturbs the balance between the proteases and the inhibitors, and eventually leads to accumulation of an aberrant intermediate,/3A4 protein. Acknowledgements. This work was supported by Grants-in-Aid and a Special Grant for Clinical Investigation from the Ministry of Education, Science and Culture, Japan, and a grant from the Sasagawa Health Science Foundation. We thank Drs. M. Kameyama (Sumitomo Hospital), T. Takeda (Kyoto University) and K. Uragami (Tottori University) for useful discussion, and Dr. B.R. Das (Institute of Life Sciences, India) for collaborative work. Drs. M. Ogawa, F. Udaka, K. Hara and R. Matsumoto (Kyoto University-affiliated hospitals) are gratefully acknowledged for providing us with brain samples.
REFERENCES 1 Anderson, J.P., Esch, F.S., Keim, P.S., Sambamurti, K., Lieberburg, I. and Robakis, N.K., Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 Cells, Neurosci. Lett., 128 (1991) 126-128. 2 Breitbart, R.E., Andreadis, A. and NadaI-Ginard, B., Alternative splicing: a ubiquitous mechanism for the generation of multiple protein isoforms from single genes, Annu. Rev. Biochem., 56 (1987) 467-495. 3 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease, Biochemistry, 18 (1979) 5294-5299.
4 De Sauvage, F. and Octave, J.-N., A novel mRNA of the A4 amyloid precursor gene coding for a possibly secreted protein, Science, 245 (1989) 651-653, 5 Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A.R., Oltersdorf, T., McClure, D. and Ward, P.J., Cleavage of amyloid fl peptide during constitutive processing of its precursor, Science, 248 (1990) 1122-1124. 6 Glenner, G.G. and Wong, C.W., Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein, Biochem. Biophys. Res. Commun., 120 (1984) 885-890. 7 Golde, T.E., Estus, S., Usiak, M., Younkin, L.H. and Younkin, S.G., Expression of fl amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer's disease using PCR, Neuron, 4 (1990) 253-267. 8 Ishiura, S., Tsukahara, T., Tabira; T. and Sugita, H., Putative N-terminal splitting enzyme of amyloid A4 peptides is the ~multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells, FEBS Lett., 257 (1989) 388-392. 9 Ishiura, S., Tsukahara, T., Tabira, T., Shimizu, T.I~Arahata, K: and Sugita, H., Identification of a putative amyloid A4-generating enzyme as a prolyl endopeptidase, FEBS Left., 260 (1990) 131134. 10 Johnson, S.A., Pasinetti, G.M., May, P.C., Ponte, P.A., Cordell, B. and Finch, C.E., Selective reduction of mRNA for the flamyloid precursor protein that lacks a Kunitz-type protease inhibitor motif in cortex from AIzheimer brains, Exp. Neurol., 102 (1988) 264-268. 11 Johnson, S.A., Rogers, J. and Finch, C.E., APP-695 transcript prevalence is selectively reduced during Alzheimer's disease in cortex and hippocampus but not in cerebellum, Neurobiol. Aging, 10 (1989) 267-272. 12 Johnson, S.A., McNeill, T., Cordell, B. and Finch, C.E., Relation of neuronal APP-751/APP-695 mRNA ratio and neuritic plaque density in Alzheimer's disease, Science, 248 (1990) 854-857. 13 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and MiillerHill, B., The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325 (1987) 733-736. 14 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H., Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity, Nature, 331 (1988) 530-532. 15 Kitaguchi, N., Takahashi, Y., Oishi, K., Shiojiri, S, Tokushima, Y., Utsunomiya, T. and Ito, H., Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of AIzheimer's disease: different properties compared with protease nexin I, Biochim. Biophys. Acta, 1038 (1990) 105-113. 16 K6nig, G., Masters, C.L. and Beyreuther, K., Retinoic acid induced differentiated neuroblastoma cells show increased expression of the A4 amyloid gene of Alzheimer's disease and an altered splicing pattern, FEBS Lett., 269 (1990) 305-310. 17 K6nig, G., Salbaum, J.M., Wiestler, O., Lang, W., Schmitt, H.P., Masters, C.L. and Beyreuther, K., Alternative splicing of the flA4 amyloid gene of Alzheimer's disease in cortex of control and Alzheimer's disease patients, Mol. Brain Res., 9 (1991) 259-262. 18 Koo, E.H., Sisodia, S.S., Cork, L.C., Unterbeck, A., Bayney, R.M. and Price, D.L., Differential expression of amyloid precursor protein mRNAs in cases of Alzheimer's disease and in aged nonhuman primates, Neuron, 2 (1990) 97-104. 19 Kuro-o, M., Nagai, R., Nakahara, K., Katoh, H., Tsai, R.-C., Tsuchimochi, H., Yazaki, Y., Ohkubo, A. and Takaku, F., cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis, J. Biol. Chem., 266 (1991) 3768-3773. 20 Lemaire, H.G., Salbaum, J.M., Multhaup, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K. and Miiller-Hill, B., The preA4695 precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons, Nucl. Acids Res., 17 (1989) 517-522. 21 Magnuson, V.L., Young, M., Schattenberg, D.G., Mancini, M.A., Chen, D., Steffensen, B. and Klebe, R.J., The alternative splicing of fibronectin pre-mRNA is altered during aging and in response to growth factors, J. Biol. Chem., 266 (1991) 14654-14662.
310 22 Masters, C.L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R.N. and Beyreuther, K., Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels, EMBO Z, 4 (1985) 2757-2763. 23 Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K., Amyloid plaque core protein in Alzheimer disease and Down syndrome, Proc. Natl. Acad. Sci. USA, 82 (1985) 4245-4249. 24 McKahnn, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease, Neurology, 34 (1984) 939-944. 25 Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promotor, Nucl. Acids Res., 12 (1984) 7035-7058. 26 Mountjoy, C.Q., Tomlinson, B.E. and Gibson, P.H., Amyloid and senile plaques and cerebral blood vessels. A semi-quantitative investigation of a possible relationship, 3~ Neurol. Sci., 57 (1982) 89-103. 27 Neve, R.L., Finch, E.A. and Dawes, L.R., Expression of the Alzheimer amyloid precursor gene transcripts in the human brain, Neuron, 1 (1988) 669-677. 28 Neve, R.L., Rogers, J. and Higgins, G.A., The Alzheimer amyloid precursor-related transcript lacking the/3/A4 sequence is specifically increased in Alzheimer's disease brain, Neuron, 5 (1990) 329-338. 29 Ohyanagi, Y., Takahashi, K., Kamegai, M. and Tabira, T., Developmental and differential expression of beta amyloid protein precursor mRNAs in mouse brain, Biochem. Biophys. Res. Commun., 167 (1990) 54-60. 30 Oppenheimer, D.R., Disease of the basal ganglia, cerebellum and motor neurons. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen (Eds.), Gleenfield's Neuropathology, 4th edn., Edward Arnold, London, 1984, pp. 699-747. 31 Oyama, F., Shimada, H., Oyama, R., Titani, K. and Ihara, Y., Differential expression of/3 amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP-751 and four-repeat tau, J. Neuropathol. Exp. Neurol., 50 (1991) 560-578. 32 Pagani, F., Zagato, L., Vergani, C., Casari, G., Sidoli, A. and Baralle, F.E., Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat, J. Cell Biol., 113 (1991) 1223-1229. 33 Palmert, M.R., Golde, T.E., Cohen, M.L., Kovacs, D.M., Tanzi, R.E., Gusella, J.F., Usiak, M.F., Younkin, L.H. and Younkin, S.G., Amyloid protein precursor messenger RNAs: differential expression in Alzheimer's disease, Science, 241 (1988) 1080-1084. 34 Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., Fuller, F. and Cordell, B., A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors, Nature, 331 (1988) 525-527. 35 Quon, D., Wang, Y., Catalano, R., Scardina, J.M., Murakami. K. and Cordell, B., Formation of /3-amyloid protein deposits in brains of transgenic mice, Nature, 352 (1991) 239-241.
36 Sisodia, S.S., Koo, E.H., Beyreuther, K., Unterbeck, A. and Price, D.L., Evidence that /3-amyloid protein in Alzheimer's disease is not derived by normal processing, Science, 248 (1990) 492-495. 37 Small, D.H., Moir, R.D., Fuller, S.J., Michaelson, S., Bush, A.I., Li, Q.-X., Milward, E., Hilbich, C., Weidemann, A., Beyreuther, K. and Masters, C.L., A protease activity associated with acetylcholinesterase releases the membrane-bound form of the amyloid protein precursor of Alzheimer's disease, Biochemistry, 30 (1991) 10795-10799. 38 Spillantini, M.G., Hunt, S.P., Ulrich, J. and Goedert, M., Expression and cellular localization of amyloid /3-protein precursor transcripts in normal human brain and in Alzheimer's disease, Mol. Brain Res., 6 (1989) 143-150. 39 St. George-Hyslop, P.H., Haines, J.L., Farrer, L.A., Polinsky, R., Van Broeckhoven, C., Goate, A., McLachlan, D.R.C., Orr, H., Bruni, A.C., Sorbi, S., Rainero, 1., Foncin, J.-F., Pollen, D., Cantu, J-M., Tupler, R., Voskresenskaya, N., Mayeux, R., Growdon, J., Fried, V.A., Myers, R.H., Nee, L., Backhovens, H., Martin, J-J., Rossor, M., Owen, M.J., Mullan, M., Percy, M.E., Karlinsky, H., Rich, S., Heston, L., Montesi, M., Mortilla, M., Nacmias, N., Gusella, J.F., Hardy, J.A. and other members of the FAD Collaborative Study Group, Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder, Nature, 347 (1990) 194-197. 40 Tanaka, S., Nakamura, S., Ueda, K., Kameyama, M., Shiojiri, S., Takahashi, Y., Kitaguchi, N. and Ito, H., Three types of amyloid protein precursor mRNA in human brain: their differential expression in Alzheimer's disease, Biochem. Biophys. Res. Commun., 157 (1988) 472-479. 41 Tanaka, S., Shiojiri, S., Takahashi, Y., Kitaguchi, N., lto, H., Kameyama, M., Kimura, J., Nakamura, S. and Ueda, K., Tissuespecific expression of three types of/3-protein precursor mRNA: enhancement of protease inhibitor-harboring types in Alzheimer's disease brain, Biochern. Biophys. Res. Commun., 165 (1989) 14061414. 42 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J.F. and Neve, R.L., Protease inhibitor domain encoded by an amytoid protein precursor mRNA associated with Alzheimer's disease, Nature, 331 (1988) 528-530. 43 Tomlinson, B.E. and Corsellis, J.A.N., Ageing and the dementias. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen (Eds.), Gleenfield's Neuropathology, 4th edn., Edward Arnold, London, 1984, pp. 951-1025. 44 Whitson, J.S., Selkoe, D.J. and Cotman, C.W., Amyloid /3 protein enhances the survival of hippocampal neurons in vitro, Science, 243 (1989) 1488-1490. 45 Wong, C.W., Quaranta, V. and Glenner, G.G., Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related, Proc. Natl. Acad. Sci. USA, 82 (1985) 8729-8732. 46 Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L. and Neve, R.L., Neurotoxicity of a fragment of the amyloid precursor associated with Alzheimer's disease, Science, 245 (1989) 417-420. 47 Yankner, B.A., Duffy, L.K. and Kirschner, D.A., Neurotrophic and neurotoxic effects of amyloid/3 protein: reversal by tachykinin neuropeptides, Science, 250 (1990) 279-282. 48 Yoshikai, S., Sasaki, H., Doh-ura, K., Furuya, H. and Sakaki, Y., Genomic organization of the human amyloid beta-protein precursor gene, Gene, 87 (1990) 257-263.
