Accumulation of Amyloid β Protein in Transgenic Mice

Neurobiology of Aging, Vol. 19, No. 1S, pp. S59 –S63, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00

PII:S0197-4580(98)00043-8

Accumulation of Amyloid b Protein in Transgenic Mice M. SHOJI,*1 T. KAWARABAYASHI,* M. SATO,† A. SASAKI,‡ E. MATSUBARA,* Y. IGETA,* M. KANAI,* Y. TOMIDOKORO,* M. SHIZUKA,* K. ISHIGURO,* Y. HARIGAYA,* K. OKAMOTO,* AND S. HIRAI§ *Department of Neurology and ‡Pathology, Gunma University School of Medicine, 3-39-15 Showamachi, Maebashi, Gunma 371, Japan †Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa 259-11, Japan §Tokyo Metropolitan Neurological Hospital, 2-6-1 Musashidai, Futyu, Tokyo 183, Japan SHOJI, M., T. KAWARABAYASHI, M. SATO, A. SASAKI, E. MATSUBARA, Y. IGETA, M. KANAI, Y. TOMIDOKORO, M. SHIZUKA, K. ISHIGURO, Y. HARIGAYA, K. OKAMOTO, AND S. HIRAI. Accumulation of amyloid b protein in transgenic mice. NEUROBIOL AGING 19(1S) S59 –S63, 1998.—Carboxyl-terminal fragments of b amyloid precursor protein (bAPP) were expressed in mice under the transcriptional control of an ubiquitous promoter system, based upon a chicken b-actin (bA) promoter combined with cytomegalovirus (CMV) enhancer to obtain a systemic overproduction of amyloid b protein (Ab). Three transgene constructs were designed to encode signal peptide and carboxyl-terminal 99 amino acid residues to bAPP (NOR-b), methionine and C-terminal 103 amino acid residues of bAPP (DNOR-b), and methionine and C-terminal 103 amino acid residues with KM-NL substitution of bAPP (DNL-b). Although the transcriptional mRNA level and post-translational protein level from transgenes showed the same expression pattern, both the expression of Ab and distribution of Ab deposits were completely different among these strains. In NOR-b mice, considerable amounts of Ab were detected in plasma and Ab deposits were observed in the pancreas. Brain Ab deposits and small amounts of plasma Ab were recognized in DNL-b. These findings indicate that tissue specific processing and transgene constructs are major facters to determine the distribution of Ab deposits. © 1998 Elsevier Science Inc. Ab

bAPP

Transgenic mice

Tissue specific processing

ALZHEIMER’S disease (AD), one of the most devastating brain diseases, is a medical, sociological, and economic problem in modern society. About 600,000 AD patients and 60 AD families are present in Japan. The progress of AD research in the last decade identified two major accumulated materials, amyloid b protein (Ab) and hyperphosphorylated tau. The deposition of Ab, a ;4-kD peptide (7,18) derived from the Ab precursor (bAPP) of ;120 kD (15), is a specific, early event in the development of AD. Then after several decades, neurofibrillary tangles and dementia appear. Genetic studies have revealed some cosegregated genes with familial AD. They are bAPP (8), presenilin 1 (21), 2 (17), and a major risk factor gene ApoE4 (3). Recent studies have suggested the meaning of mutation of bAPP and presenilins are overproduction of Ab1-42 (1,2,4,23). Based on these findings, Ab1-42 is considered to be a common initiating factor of AD (9,14,20). This is a reason to make AD model mice expressing considerable amounts of Ab to analyze how Ab accumulates and progresses, and finally how to prevent this pathological development (6,11). Because the native bAPP is ubiquitously expressed, and the cultured cells transfected with carboxyl-terminal fragment (CTF) of bAPP (5) release Ab effectively (12,22), we generated transgenic mouse lines overexpressing CTF under the transcriptional control of an ubiquitous promoter system, based upon a chicken b-actin (bA) promoter combined with cytomegalovirus (CMV) 1

Amyloid deposits

enhancer to obtain a possible overproduction of Ab (16). Three transgene constructs were designed to code signal peptide and C-terminal 99 amino acid residues of bAPP (NOR-b) (16), methionine plus C-terminal 103 amino acid residues of bAPP (DNOR-b), and methionine plus C-terminal 103 amino acid residues with KM-NL substitution (19) of bAPP (DNL-b) for analyzing Ab production and accumulation from expressed proteins in vivo. METHODS

The NOR-b gene construct was inserted into the EcoRI site of the third exon of the rabbit b-globin gene in pBsCAG-2. The transgene consisted of a ;2.35-kb SalI-BamHI fragment containing bA, the first intron of the chicken b-actin gene, NORb gene, and a part of the rabbit b-globin gene (16). The DNOR-b gene encoding methionine 1 C-terminal 103 amino acid residues of bAPP and DNL-b gene encoding methionine 1 C-terminal 103 amino acid residues with KM-NL substitution at bAPP595/596 were generated by using PCR from cDNA of bAPP695 with primer sets of 59-GGTCTAGAGATGGAAGTGAAGATG-39, 59GGTCTAGAGATGGAAGTGAATCTGGAT-39, and 59GGAGATCTCGATCAAGACGTA-39. These gene constructs were inserted into the XbaI site of the third exon of the rabbit b-globin gene in pBsCAG-2. Approximately 2000 copies of the

To whom correspondence should be addressed.

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respective transgene were micro-injected into the pronuclei of fertilized B6C3F1 3 C57BL/6N hybrid eggs. Respective high expression Fo lines were back crossed to C3H mice for analysis. Southern blot analysis of tail-derived DNA and Northern blot analysis from various transgenic organs were performed to select high expression lines and to evaluate systemic expression of mRNA from the transgene. The antiserum Saeko to carboxylterminal of bAPP was used to detect expression proteins in Western blot. The anti-Ab1-40 antibody (Mizuho), 4G8, and 6E10 were used to detect Ab deposits (10,16). Carboxyl-terminal ends of deposited Ab species were also examined by using site-specific antibodies, S40 to Ab ending at 40V, S42 to Ab ending at 42A, and S43 to Ab ending at 43T (13). The antibody, F4/80 (Serotec, Oxford, UK), was used to detect mature mouse macrophages (16). Tissue samples were fixed in 4% paraformaldehyde with 0.1 M phosphate buffer (pH 7.6). Paraffin sections were immunostained using primary antibodies (1:500) and horseradish peroxidaseconjugated second antibody (1:200; Tago, CA). Immunoreactivity was visualized by incubation with 0.03% 3, 39-diaminobenzidine, 0.065% sodium azide, and 0.02% H2O2. Methylgreen was used for nuclear staining. Tissue samples were immersed in the fixative (2.5% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4) for 4 h and washed several times in 0.1 M phosphate buffer (pH 7.4) containing 7% sucrose. They were then post-fixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in Quetol 812 (Nisshin EM, Japan). Ultrathin sections were stained with uranyl acetate and lead acetate prior to observation with an electron microscope. Within high expression lines, 4 founders and their progeny (F1-F4) aged 4 weeks to 24 months in each strain were examined. RESULTS AND DISCUSSION

Although there were no consistent Ab deposits in the brain, NOR-b mice showed extra neural Ab deposits in the pancreas. Immunostaining with antibody to Ab, Mizuho showed intracellular accumulation of Ab in acinar cells of the pancreas. These Ab deposits appeared at 4 months of age and progressed with aging. Hematoxylineosin staining showed age-related degeneration of acinar cells. Macrophage staining showed reactive infiltration of macrophages accompanied with aging. Electron microscopic (EM) examination showed deposits of intracellular fibrils, degenerated lysosomes of acinar cells and cytoplasm of infiltrated macrophages. Some acinar cells showed severe degeneration under EM study. However, little amounts of Ab fibrils were recognized in extracellular spaces. Antibodies to Ctermini of Ab showed that early components of intracellular Ab deposits in the pancreas were Ab ending at position 42 (Ab42). Ab42 appeared at 4 months of age, Ab ending at position 40 and 43 accumulated after 8 months of age. Based on these findings (16), we consider that intracellular Ab deposits in the pancreas consisted mainly of Ab42. Ab deposits in the pancreas were quite similar to Ab deposits in AD brains (9,10). Another sensitive method for detection of Ab by ELISA showed that considerable amounts of Ab were accumulated in the brain, kidney, and pancreas in NOR-b (16,23). In plasma, both 20 pmol/mL of Ab1-40 and 51 pmol/mL of Ab1-42 were detected. However, no Ab deposits appeared in the parenchyma and blood vessels in the brain. These findings indicate that NOR-b mice actually produce and release Ab and that tissue-specific processing determines the distribution and level of Ab deposits in NOR-b mice. To examine the difference in distribution and amounts of expression proteins, high expression lines of DNOR-b and DNL-b were also analyzed. In RNA samples from various transgenic

FIG. 1. Northern blot analysis. Lane 1, 2, and 3, control non-transgenic mice; Lane 4, DNOR-b mice; Lane 5 and 6, DNL-b mice; Lane 7 and 8, NOR-b mice. RNA was isolated from liver of each lines using guanidinium thiocyanate (3). Twenty mg of total RNA per lane was electrophoresed through a 1.1% agarose-formaldehyde gel, followed by capillary transfer to nylon filter membrane (Pall Bio Support Division). The blots were hybridized with radiolabeled NOR-b at 42°C in 5 3 SSC, 50% formamide, 5 3 Denhardt’s, 0.15% SDS, 100 mg/mL salmon sperm DNA for 16 –18 h using standard technique. Filters were washed once in 0.1 3 SSC/0.1% SDS at 56°C for 30 min. prior to autoradiography.

organs, the predicted 1.0 kb transgenic mRNA was invariably detected, although the amount varied widely from tissue to tissue. The patterns of transgene mRNA level were almost equal in all organs among the 3 strains, NOR-b, DNOR-b, and DNL-b at 8 months of age (Fig. 1). Expression of full length endogenous bAPP varied from tissue to tissue with particularly high expression in the brain. However, there were no differences among the 3 transgenic strains and non-transgenic mice. Analysis of transgenederived proteins by immunoblot using Saeko showed a quite interesting expression pattern of protein. These transgenes expressed 11.4 kD C-terminal fragment of bAPP (CTF) in NOR-b and 11.8 kD CTF in DNOR-b and DNL-b in the brain, lung, kidney, pancreas, heart, intestine, and muscles. Although the amounts of expressed proteins were different in various organs from one strain, the same pattern of expressed CTFs was observed among NOR-b, DNOR-b, and DNL-b. Large amounts of 11.4 kD/11.8 kD CTF were accumulated in the liver and the pancreas in these transgenic strains (Fig. 2). Thus, chicken b-actin promoter

FIG. 2. Analysis of transgene derived proteins by immunoblot using Saeko. These transgenes expressed 11.4 kD C-terminal fragment of bAPP (CTF) in NOR-b and 11.8 kD CTF in DNOR-b and DNL-b in the brain, lung, kidney, pancreas, heart, intestine, and muscles. B, brain; L, liver; K, kidney; P, pancreas; H, heart; I, intestine; M, muscle.

Ab DEPOSITS IN TRANSGENIC MICE

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FIG. 3. Immunostaining of pancreas and the brain of each line by antibody S42 (3 650). Only NOR-b mice showed Ab deposits in the pancreas among the 3 strains. Ab immunostaining of DNL-b mice showed intra-neural Ab deposits only in the brains.

combined with cytomegalovirus enhancer expressed very similar patterns and levels of both protein and mRNA from the transgene among these three different strains. Furthermore, no significant differences were observed among founders of each strain. These findings suggest that the differences in amounts of CTF are dependent on organs, not on transgene constructs or insertion sites. Post-translational tissue-specific processing of expression protein may cause these differences. However, amounts of Ab in plasma were completely different among three strains. There are 20 pmol/mL of Ab1-40 and 51 pmol/mL of Ab1-42 in NOR-b, 0.4 pmol/mL of Ab1-40 and 0.1 pmol/mL of Ab1-42 in DNOR-b, and 4.1 pmol/mL of Ab1-40 and 18.8 pmol/mL of Ab1-42 in DNL-b. Fifty-one pmol/mL of Ab1-42 is 500-fold more than in human plasma. Considerable amounts of plasma Ab1-42 did not cause any amyloid angiopathy or brain amyloid deposits. These findings suggest that 1) Ab is actually produced and released in these three strains, and 2) the amount of released Ab is dependent on the presence of signal sequence or KM-NL mutations in the transgene construct. Ab immunostaining showed more interesting findings. Only NOR-b mice showed age-related Ab deposits in the pancreas among the three strains. A few small granules labeled with Mizuho appeared in some acinar cells at 3 weeks of age, then, the number and size of these Ab-immunoreactive granules increased with aging from 4 months after birth to 24 months of age. Abimmunoreactive interstitial macrophages were detected at 8 months of age. The number of these cells filled with Ab also

increased with age and their clusters occupied several parts of the pancreas. Such Ab deposits were not detected in the pancreas of non-transgenic mice at 20 months of age. Although large amounts of CTF were recognized in the pancreas, these Ab deposits in the pancreas were not observed in either DNOR-b or DNL-b mice. No Ab deposits were observed in any organs including brains of DNOR-b. However, Ab immunostaining of DNL-b mice showed intra-neural Ab deposits only in the brains from 8 months of age. Ab deposits were recognized in the cytoplasm of pyramidal cells in the hippocampus and cerebral cortex. The number and size increased with age. Using more site-specific antibodies, the brain of DNL-b was examined in detail. These Ab deposits were labeled by S40, S42, S43, and 4G8 immunostaining. Immunoelectron microscopy revealed amorphous substances labeled by Mizuho and S42 in lysosomes of neurons (Fig. 3). These findings showed that Ab deposits in the brain of DNL-b consisted mainly of Ab ending at position 42(43), as shown in the AD brains. No pancreatic Ab deposits were observed in DNL-b any age. Immunoblot study of SDS-resistant formic acid soluble fractions from various organs from NOR-b, DNOR-b, and DNL-b confirmed that 4 kD Ab labeled by 6E10 was accumulated in the pancreas of NOR-b and in the brain of DNL-b. These findings also confirmed biochemically that Ab immunoreactivity in pancreas of NOR-b and the brain of DNL-b were Ab deposits. These findings from experiments using three different transgenic mice are summarized in Table 1. Expression patterns of CTF in organs were quite similar among the three strains. However, the

TABLE 1 SUMMARY OF THREE STRAINS OF TRANSGENIC MICE Gene construct Strain

Signal peptide

NOR-b DNOR-b DNL-b

1 2 2

Expression protein

CTF

NL/KM mutation

Molecular weight

Brain

Liver

C99 M1C103 M1C103

2 2 1

11.4 kD 11.8 kD 11.8 kD

1 1 1

11 11 11

Ab deposits Pancreas

Plasma (Ab)

Brain

Pancreas

111 11 11

111 6 1

2 2 1

111 2 2

CTF, carboxyl-terminal fragment of bAPP; C99, carboxyl-terminal 99 amino acids residues of bAPP; M, methionine; C103, carboxyl-terminal 103 amino acids residues of bAPP.

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amounts of plasma Ab, i.e., the Ab production activities, were completely different. Although large amounts of CTF were observed in the liver and the pancreas in all strains, Ab accumulated only in the pancreas of NOR-b. Furthermore, brain Ab deposits were observed only in DNL-b mice although the levels of CTF expressed in the brain were not different among three transgenic strains. Because the promoter and translational level of each transgene were very similar, tissue specific post-translational processing for generating and degrading Ab were critically important factors of Ab accumulation in an organ in addition to the expression rate of bAPP. Gene constructs used here are considered to be another important factor for distribution of Ab deposits. Signal peptide may be related with release of Ab into blood and Ab accumulation in pancreas. The presence of KM-NL mutations of bAPP may work as a signal for brain Ab deposits. Overproduction of Ab, especially Ab1-42, is considered to be a critically

important factor for acceleration of Ab deposits. However, our study indicates some additional factors. Mutation of bAPP is considered to be important not only for the level of Ab production but also for the distribution of Ab deposits. Furthermore, tissue specific processing and degrading system of soluble Ab may be more important factors to determine the distribution and amounts of Ab deposits. We hope that these findings offer some insights to clarify the mechanism of Ab deposits in sporadic AD patients. ACKNOWLEDGEMENTS

We thank Dr. Cris Eckman and Dr. Steve Younkin for bAPP gene and ELISA. This work was supported by the Life Science Foundation, Mochida Memorial Foundation, the Univers Foundation, the Sasakawa Health Science Foundation, the Primary Amyloidosis Research Committee, and the Longevity Science Committee of the Ministry of Health and Welfare of Japan and the Ministry of Education, Science, and Culture of Japan.

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