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Division of Laboratory Animal Resources and
Division of Neurotoxicology, Korea FDA, National Institute of Toxicological Research, Seoul 122–704;
* Collage of Pharmacy, Chungbuk National University, Cheongju 361–763; and
Department of Biology, Chungnam National University, Taejon 305–704, Korea
1Correspondence: Division of Laboratory Animal Resources, Korea FDA, National Institute of Toxicological Research, 5 Nokbun-dong Eunpyng-ku, Seoul 122–704, Korea. E-mail: kimyongkyu{at}hanmail.net
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretase. An increased production of Aß-42 by mutation of PS2 genes promotes caspase expression and is associated with the Cox-2 found in the brain of AD patients. To address this question in vivo, we expressed the human mutant PS2 (hPS2m) (N141I) as well as wild PS2 (hPS2w) as a control in transgenic (Tg) mice under control of the neuron-specific enolase (NSE) promoter. Water maze tests were used to demonstrate the behavioral defect; dot blot, Western blot, and immunohistochemical analyses were performed on the brain with the hPS2, Aß-42, caspase-3, and Cox-2 antibody. We concluded that 1) Tg mice showed a behavioral dysfunction in the water maze test, 2) levels of hPS2, Aß-42, caspase-3, and Cox-2 expression were modulated in the brains of both Tg mice, 3) dense staining with antibody to hPS2, Aß-42, caspase-3, and Cox-2 was visible in the brains of Tg mice compared with age-matched control mice, and 4) distinguishable AD phenotypes between hPS2w- and hPS2m-Tg mice did not appear. These results suggest that an elevation of Aß-42 by overexpression of hPS2 and mutation of hPS2m might induce the behavioral deficit and caspase-3 and Cox-2 induction, which could be useful in the therapeutic testing of compounds to have considerable clinical effects.—Hwang, D. Y., Chae, K. R., Kang, T. S., Hwang, J. H., Lim, C. H., Kang, H. K., Goo, J. S., Lee, M. R., Lim, H. J., Min, S. H., Cho, J. Y., Hong, J. T., Song, C. W., Paik, S. G., Cho, J. S., Kim, Y. K. Alterations in behavior, amyloid ß-42, caspase-3, and Cox-2 in mutant PS2 transgenic mouse model of Alzheimer’s disease.
Key Words: AD • PS2 • Aß-42 • amyloid precursor protein
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-secretase enzymes. An 
-secretase cleaves the middle of the Aß region containing the first 16 amino acids of the Aß (sAPP), releasing a secreted ectodomain. A ß-secretase cleaves between Met 671 and Asp 672, producing the amino-terminal end of the Aß and releasing a secreted ectodomain composed of a truncated form ending with Met 671 (sAPPß), the substrate of -secretase. Recent results suggest presenilin-1 (PS-1) and presenilin-2 (PS-2) are components of a multiprotein -secretase complex (1)
necessary for production of the neurotoxic Aß.
At least four gene mutations are linked to the early onset of AD including the ß-amyloid precursor protein (ß-APP) (2)
, PS-1 (3)
, PS-2 (4)
, and apolipoprotein E type 4 (APOE-E4) (5
, 6)
. Among these, the mutated PS genes were observed in the larger proportion of patients with early onset of AD. Thirty families (60%) have missense mutations (3
, 7)
or amino acid deletion (8)
in the PS-1 gene. Two mutations in the PS2 gene have been described in eight families resulting in the substitution of Asn-141 by Ile (N141I) (9)
, and another missense mutation (M239V) (4)
. Both genes encode the sixth and seventh transmembrane proteins and show a high degree of homology at the amino acid level (67%) by sequence comparison (9)
. Encoded proteins are located predominantly in the endoplasmic reticulum and less abundantly in the Golgi apparatus (10
11
12)
. They share similar structural features: six to eight times in the amino- and carboxyl-terminal domains (13)
;the large loop spanning the putative sixth and seventh transmembrane domains contained within the cytoplasm. Their loop and amino terminus are regions of significant sequence divergence between the PS-1 and PS-2 proteins. Proteolytic cleavage of the PS-1 and PS-2 are endoproteolytically processed in vivo and cell transfectants to yield 27–35 kDa amino-terminal and 15–24 kDa carboxyl-terminal fragments (14
15
16)
.
Missense mutations in PS genes result in an increased Aß-42 level in the plasma of AD patients, caused by a shifting of two amino acids in -secretase’s preferred cleavage site in the APP sequence (17)
. Since the PS proteins are the -secretases responsible for intramembranous processing of APP (18)
, it results in an increase of abnormally structured Aß-42 that can be neurotoxic in vitro and in vivo (19)
. The mechanisms of their neurotoxicity are still unknown. Increased Aß might induce apoptotic activity by interacting with neuronal receptors, including the receptor for advanced glycation end-products (RAGE) (20)
, the p75 neurotrophin receptor (21)
, and the APP (22)
. Therefore, enhanced Aß deposition may be correlated with expression of the caspase-3 and Cox-2 found in AD patients.
In this study, we produced the Tg mouse model expressing hPS2m (N1411) and hPS2w under control of the NSE promoter. These mice showed a behavioral defect in the water maze test. These mice were then used to examine whether deposition of the Aß is increased in hPS2w and hPS2m mice and its association with expressions of caspase-3 and Cox-2. Our study shows an elevation of Aß-42 and caspase-3 in the cortex and hippocampus region of hPS2m and hPS2w-Tg mice. In parallel, we found that enhanced expression of Cox-2 in dentate gyrus, CA1, and CA3 of the hippocampus is almost colocalized with that of Aß-42 deposition.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and the hPS2m (N141I, Volga German Families). Each SacII/XbaI fragment of hPS2w and hPS2m was inserted in pUHD10–3 (14)
. The pNSE-CAT and pUHD10–3 were gifts from Dr. J. Gregor Sutcliffe at the Research Institute of Scripps Clinic and Dr. Tae-Wan Kim at Columbia University, respectively. The hPS2w and hPS2m coding sequences are linked to the SV40 polyadenylation signals downstream.
Production of Tg
The pNSE-hPS2w and pNSE-hPS2m were digested with SwaI and AflIII to remove prokaryotic sequences, then diluted to 4 ng/µl. Each linear NSE-PS2w and NSE-PS2m fragment was microinjected into the male pronucleus of fertilized mice eggs. The egg were obtained by mating the female BDF1 with male BDF1 mice. Injected eggs were transferred to the oviducts of pseudopregnant ICR recipient females on day 1. Five males and three females of newborn mice were obtained, one of each (2348, 2346) carried the hPS2w transgene. In the hPS2m-Tg mice, two females (1480 and 1146) of nine offspring were identified as Tg mice. Tg lines and age-matched control mice were handled in an accredited Korea FDA animal facility in accordance with the AAALAC International Animal Care policies. Mice were housed in cages under a strict light cycle (light on at 06:00 and off at 18:00). All mice were given a standard irradiated chow diet (Purina Mills, St. Louis, MO) ad libitum. The mice were maintained in a specified pathogen-free state. All pedigrees were hemizygous for their transgene.
Southern blotting
Genomic DNA was prepared from the tails of 4-wk-old founder mice and the transgene was detected by Southern blot analysis of EcoRI-digested tail DNA. After electrophoresis, DNA was transferred overnight to nylon membranes in 10x SSC in accordance with the manufacturer’s instructions. Membranes were then immersed in 0.4 N NaOH, neutralized with 0.2 M Tris-HCl (pH7.5), 2x SSC, and allowed to air dry. Each membrane was prehybridized at 65°C for 2 h in a hybridization buffer. The 32P-labeled PS2 probe was added to the membranes and hybridization was allowed to proceed at 65°C for 18 h. The membrane was then washed as follows; 15 min, 2x SSC/0.2% SDS at room temperature (once); 15 min, 1x SSC/0.2% SDS at 65°C (3x), and 30 min, 0.5x SSC/0.2% SDS at 65°C (3x). Filters were then exposed to Kodak XAR film at -70°C. The 558 bp PS2-specific probe was prepared by digestion with ApaI enzyme. Fragments were separated by agarose gel electrophoresis and purified by passage through a Jetsorb (Genomed 110150).
PCR analysis
The transgenes were identified by DNA-PCR analysis from genomic DNA isolated from the tails of 4-wk-old founder mice. The hPS2w and hPS2m genes were synthesized using sense primer (5'-GAGGA AGAAG TGTGT GATGA G-3) and antisense primer (5'-CACGA TGACG CTGAT CATGA TG-3), with complementary hPS2w and hPS2m genes ranging from 395 to 416 and 817 to 796 nucleotides as the DNA template. After 25 cycles of amplification, the levels of hPS2w and hPS2m products (422 bp) were quantified using a Kodak Electrophoresis Documentation and Analysis System 120 on 1% agarose gels. For RT-PCR analysis, each tissue was frozen with liquid nitrogen. Frozen tissue was chopped with scissors and homogenized in the RNAzol B solution (Tet-Test Inc. CS104). Isolated RNA was then quantified by UV spectroscopy. To examine the expression of transgenes, RT-PCR was performed using 5 µg of total RNA from each tissue; 500 ng of oligo-dT primer [Gibco BRL(18418–012)] was annealed at 70°C for 10 min. Complementary DNA serving as template for further amplification was synthesized by addition of dATP, dCTP, dGTP, dTTP, and 200 unit of reverse transcriptase. Thereafter, 10 pmol of sense and antisense primers was added and the reaction mixture subjected to 28 cycles of amplification. Amplification was carried out on a Perkin-Elmer Thermal Cycler using the following cycle: 30 s, 94°C; 30 s, 62°C; 45 s, 72°C. In each case, minus RT controls were included to distinguish between DNA and RNA products. RT-PCR using specific primer for ß-actin was performed to ensure the integrity of the RNA. The primers of hPS2w and hPS2m used for PCR: sense primer, 5'-AGAAC ACTGC CCAGT GAAGA AG-3' and antisense primer, 5'-CACGA TGACG CTGAT CATGA TG-3', corresponding to the nucleotides 492 to 513 and 796 to 817 of the hPS2 gene. The sequence of ß-actin sense primer was 5'-TGGAA TCCTG TGGCA TCCAT GAAAC-3' and the sequence of their antisense primer was 5'-TAAAA CGCAG CTCAG TAACA GTCCG-3'. The experiment was repeated multiple times, and the relative difference in RNA quantity was assured by obtaining three reproducible results.
Immunohistochemistry
Tg and age-matched mice were perfused as described (24)
. After perfusion, brain tissue was fixed in 5% formalin at 4°C for 12 h and transferred successively to 10–20% and 30% sucrose solution. Sections (20 µm-thick) were prepared and pretreated at room temperature for 30 min, then with PBS blocking buffer containing 10% goat serum in PBS for 1 h. These sections were incubated with rabbit polyclonal anti-ß-amyloid 42 unconjugated (BioSource Int., Camarillo, CA; 2 µg per ml) and goat polyclonal anti-Cox-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100 dilution) at 4°C, overnight. Rabbit polyclonal antibodies raised against caspase-3 (Santa Cruz, CA; 1:200 dilutions) were used for the reaction of immunostaining. For hPS2 immunostaining, brain sections were reacted with rabbit polyclonal anti-hPS2 antibody (Santa Cruz; 1:200 dilution). Each complex of antigen-antibody were visualized with biotinylated secondary antibody (goat anti-rabbit) -conjugated HRP streptavidin (Zymed, San Francisco; Histostain-Plus Kit) diluted 1:1500 in PBS blocking buffer.
Dot blot analysis
Protein prepared from the brains of Tg and age-matched control mice were transferred to a nitrocellulose membrane using a Slot-Blot (Pharmacia Biotech, Alameda, CA). The membrane was incubated separately with primary rabbit polyclonal anti-ß-amyloid 42 unconjugated, 2 µg in blocking buffer, at room temperature for 3 h. It was washed in washing buffer and incubated with a secondary antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG (GenTest, Woburn, MA), at a dilution 1:1000 at room temperature for 1 h. Aß-42-specific peptide was detected by an NBT/BCIP (Sigma, St. Louis, MO). Anti-ß-amyloid 42 is able to detect Aß-42 on a Slot-Blot when used as recommended by the manufacturer. Aß-42-specific peptide was detected by an enhanced chemiluminescence substrate (Pharmacia).
Western blot Analysis
For Western blot analysis, protein (10 µg) was separated in a 10–20% gradient gel of polyacrylamide electrophoresis for 3 h and transferred to a nitrocellulose membrane using an electroblot for 2 h. Each membrane was incubated separately with a primary rabbit polyclonal anti-PS2 unconjugated, a rabbit polyclonal anti-caspase-3, and a goat polyclonal anti Cox-2 antibody in blocking buffer at room temperature for overnight. The membranes were washed in washing buffer and incubated with secondary antibody and HRP-conjugated goat anti-rabbit IgG (Zymed) at 1:1000 for 2 h at room temperature.
Water maze
Twelve-month-old Tg and age-matched control mice were used for water maze tests. These tests were performed by the SMART-CS (Panlab, Barcelona, Spain) program and equipment was placed in an experimental room with window, air-conditioning, and tables. A plastic circular pool 1.5 m in diameter was filled with water and maintained at 22–25°C. The visual field in the pool was obstructed by the addition of powdered milk. Mice were pretrained to swim to a round shape platforms (diameter 12 cm) submerged 1 cm beneath the surface of the water. Escape latency, escape distance, swimming speed, and swimming pattern of the mice were monitored by a computer program (SMART-LD) connected to a camera mounted in the ceiling directly above the pool. Habituation trials of 60 s were performed to verify their ability to swim. Mice were given five training trials during which their latency to find the hidden platform was measured for a maximum of 60 s. If the mice failed to find the platform within the maximum time, they were placed on the platform by the experimenter. The training schedule consisted of two trials a day for 5 days of testing;each trial was assessed by the mouse reaching the platform in 60 s. The second trial was given at least 5 min after the first trial, and both trials were started from an identical location. The platform location was kept constant during the period of training. After each trial, mice remained on the platform for 30 s. On the sixth day, mice were subjected to three probe trials in which they swam for 60 s with no platform present in the pool. Patterns of searching, the number of times (escape latency), the distance they swam (escape distance), and speed of swimming (velocity) to the former platform location were recorded. All trials were stored on videotape for subsequent analysis of platform crossing. The numbers of mice tested in the water maze were 6 Tg mice (3 for each hPS2m and hPS2w) and 3 control mice at 12 months of age.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B). One male (2348) and one female (2346) were identified by DNA PCR and Southern blot hybridization in the hPS2w-Tg mice (Fig. 1A
). Intensity and restriction patterns of the DNA obtained from DNA-PCR and Southern blotting were compared for Tg mice using a 32P-labeled hPS2 sequence. In this experiment, a mutant Tg line (PmHK77, hPS2m) subsequently crossed with the founder mouse (1480) onto the parental BDF1 and a wild-type Tg line (PwHK71, hPS2w) that established the founder mouse (2348) with a parental strain of BDF1 were used.
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Tissue-specific regulations of NSE-hPS2w and NSE-hPS2m expression in the Tg mice
To test whether regulation of the hPS2w and hPS2m genes are expressed under the control of NSE promoter in a brain-specific manner, tissues (anterior brain, posterior brain, heart, lung, liver, kidney, intestine, and muscle) were prepared from hPS2w and hPS2m-Tg mice and their transcript levels of PS2 were examined by RT-PCR analysis. RT-PCR analysis using specific hPS2w and hPS2m primers generates a product of 352 bp. In this experiment, the highest expression of hPS2m was observed in the anterior brain, followed by posterior brain, muscle, lung, heart, and liver (Fig. 2
B). The level of hPS2w transcripts was dramatically increased in the brain of hPS2w-Tg mice. In contrast, the mRNA product of ß-actin was almost constant in all tissues tested. The fusion gene was expressed preferentially in a brain of hPS2m transgenic mice in a brain-specific manner (Fig. 2B
). Transcript levels of hPS2w in liver, kidney, and intestine were higher than in the brain of hPS2w-Tg mice (Fig. 2A
).
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Water maze testing
To assay behavioral defects, 12-month-old Tg lines and age-matched control mice were measured for escape latency, escape distance, swimming speed, and swimming pattern using a water maze test. After training (day 5), all mice on day 6 were given three trials where they swam in the pool for 60 s with the platform removed. Escape latency, escape distance, and swimming pattern across to the former platform location were recorded. All results were significantly different for Tg vs. age-controlled control mice (Fig. 3
A, B). However, swimming speed of wild-type and mutant Tg mice was slightly lower than age-matched control mice (Fig. 3B
). We observed that behavioral defects were apparent in hPS2w and hPS2m-Tg mice.
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Expression and immunoreactivity for PS2
To characterize the PS2 expression, filters and tissue sections were stained with antibody specific to PS2. Western blot analysis showed that expression of PS2 gene in hPS2w and hPS2m-Tg mice were higher than those in age-controlled mice; the full length of PS2 protein in hPS2w and hPS2m-Tg mice were visible (Fig. 4
A). We could not detect the amino-terminal fragment (NTF) and carboxyl-terminal fragment in hPS2w and hPS2m-Tg mice, but CTF and NTF appeared in another mutant PS2 Tg line as described (25
, 26)
. It may responsible for a difference in the caspase-3 activity between our Tg and other Tg lines. In fact, our Tg lines showed a weak signal of caspase-3 compared with age-matched control mice as shown in Fig. 4
. The hPS2 antibody is appears to be most reactive with the dense in the cortex and hippocampus in Tg mice (Fig. 4B
). The smallest amount of PS2 was observed in cortex and hippocampus of the control mice (Fig. 4B
).
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Dot blot and immunoreactivity for Aß-42
After the water maze test, subsets of each group of mice were killed for Aß-42 measurement by dot blot and immunohistochemical analyses using the specific antibody to Aß-42. As shown in Fig. 5
A, Aß levels in the brains of two Tg mice were two- to threefold higher than those in brains of age-matched control mice. A signal for Aß-42 levels was observed in control mice, indicating there were age-dependent increases in Aß-42 levels in the brain of non-Tg mice. This is consistent with other reports that Aß-42 in the mutant PS2-Tg mice under the controls of chicken ß-actin and platelet-derived growth factor (PDGF) ß chain promoters has a higher level than those in wild-PS2 or age-controlled control mice (25
, 26)
and suggests that overexpression of hPS2 and mutation in hPS2 resulted in the expression at the high levels of Aß-42 in the brains of Tg mice.
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To assess the pattern of Aß deposition resulting from hPS2w and hPS2m expression, we analyzed the Aßimmunoreactivity in the hippocampus and cerebral cortex of two Tg and age-matched control mice. These regions were chosen because they are areas of prominent Aß deposition and neuronal loss that correspond to the more compact morphology in human AD patients (27
28
29)
. Brain sections derived from mice were then immunostained with Aß-42-specific antibody. Staining was widespread in all regions of the cortex of Tg mice whereas a similar region from an age-matched control mice showed relatively weaker immunoreactivity (Fig. 5B
). Many cells in dentate gyrus are more immunoreactive with packed lines in the brain of hPS2m Tg mice than in hPS2w mice. Moreover, cells in the CA1, CA2, and CA3 regions are intensively immunostained with a dense line in Tg mice, whereas these regions in the control brain show a low immunoreactivity (Fig. 5B
). Our results suggest that antibody staining is specific to cells that accumulate Aß-42 by hPS2w and hPS2m, although it is unlikely for the antibody to be binding nonspecifically in the brain of age-matched control mice.
Expression and immunoreactivity for caspase-3
Aß might drive cells into apoptosis when they are associated with several receptors activating the cell death signal pathway. Caspase-3 is involved in APP processing consistent with elevation of Aß formation in neurons of AD patients (30)
. Therefore, we examined whether caspase-3 is activated in the brains of Tg mice as it is in AD patients. As shown in Fig. 6
A, the intensity of procaspase-3 protein was seen in the brains of non-Tg mice, where it was evident as weak expression of procaspase-3 protein in the brains of hPS2w and hPS2m mice (Fig. 6A
). Although the activated form of caspase-3 was not visible (Fig. 6A
), it may result from low-level expression of caspase-3. Large areas of distribution and dense neurons in caspase-3 staining were observed in the hippocampus-dentate gyrus and the cerebral cortex (Fig. 6B
). These data were reproducibly observed in the three experiments. The results suggest that caspase-3 might mediate, or contribute to the apoptosis of neuronal cells of the AD pathogenesis.
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Expression and immunoreactivity for Cox-2
Cox-2 expression was increased in frontal cortex and hippocampus of AD patients and their immunoreactivity was correlated with the number of amyloid plaques (31
, 32)
. Cox-2 was preferentially localized in neurofibrillary tangle positive neurons (33)
. We wanted to see whether Cox-2 expression is elevated in the brains of Tg mice as found in AD brains. Western blot analysis of brain protein showed that the level of Cox-2 protein in hPS2w and hPS2m-Tg mice had actually increased 1.5- to 2-fold compared with that of non-Tg mice (Fig. 7
A). CA1 and CA3 areas and the dentate gyrus area of the hippocampus showed a dense form of the Cox-2 immunopositive neuronal cells in Tg mice relative to those in the control mice, and its pattern was identical to that with Aß-42 staining pattern. A densely packed form in the Cox-2 immunopositive staining was found in the neuron of the cortex (Fig. 7B
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and chicken ß-actin promoter (26)
, these mice showed an increased level of Aß-42 in the brain of 12-month-old Tg mice. The most important finding in this study is that hPS2w and hPS2m linked to the NSE promoter not only had significant effects on levels of PS2 and Aß-42, but also on levels of caspase-3 and Cox-2 proteins. Tg mice expressing NSE-hPS2w and NSE-hPS2m show evidence of the behavior deficit. There are several possible explanations for the evidence to the phenotypes and signals underlying AD in the Tg mice. 1) This evidence may be related to the NSE promoter activity directing target hPS2w and hPS2m to neurons of Tg mice. In hPS2w-Tg mice, brain-enriched expression with about an eightfold increase was observed when 352 bp of the NSE promoter was used, although their regulation was more highly expressed in the liver, kidney, and intestine in hPS2m-mice. Therefore, the NSE promoter may be more important in the induction of Aß-42 production than other promoters like ß-actin and PDGF when it is fused to the hPS2w or hPS2m. 2) Coupled with the NSE promoter, hPS2w and hPS2m may have a more synergic effect on Aß-42 production than the other mutated genes for AD. However, it is necessary to test the strength of promoters with other mutant genes, including human PS-1and APP mutations. 3) It is possible that the BDF1 strain we used in this study may be useful in testing for learning and memory defects and is more likely to induce cognitive deficits than other strain, such as C57BL/6J. In this study, all AD phenotypes that appeared in the hPS2w-Tg line were similar to those in hPS2m-Tg mice. An hPS2m (PmHK77) and another mutant Tg line (data not shown) were shown to alter AD’s phenotypes similarly, so it is not possible to have an insertional effect on their phenotypes in these mutant lines. We assumed that overexpression of hPS2 and mutation in hPS2m affect the processing of APP somewhat similarly to stimulate Aß-42 production.
In the water maze test, 12-month-old Tg lines had longer escape latencies when crossing to the former platform location than did age-matched control mice. These wild-type and mutant Tg mice showed an increase in escape distance taken to find the former platform location. No significant difference in swimming speed was observed between the mice. The results suggest that the behavior is likely to represent a lack of memory in finding the former platform location. Using these mice with behavior defects, we found that levels of biochemical and immunostaining for Aß-42 were significantly increased without observation of plaque formation in the cortex and hippocampus of Tg mice. In fact, the behavior defect occurred before plaque formation was common in all Tg mice that overexpressed wild-type or mutant APP, including those that did not develop amyloid plaque (34)
. Therefore, mice expressing hPS2w and hPS2m may be similar to cases found in APP-Tg mice.
Alteration in caspase-3 protein was observed in the brains of Tg mice from Western blot and immunostaining analysis. Interactions of Aß with several receptors may play an important role in the induction of caspase by the cell death signal pathway. Several receptors such as RAGE (receptor for advanced glycation end products), which can produce free radicals, and APP and p75 neurotrophin receptor, which can induce the neuronal apoptosis, are involved in the induction of caspases. Aß activates a set of immediate early genes like JNK activation and phosphorylation of c-jun through receptor-mediating apoptotic signal pathway, as in the case of those induced by trophic factor withdrawal (35)
. Finally, activated caspases result in apoptosis in AD. Our results are of interest when examining whether inhibitors of caspase-3 protect for neuronal cell death in Tg mice. These Tg lines are good models for the development of therapeutic compounds for AD patients.
Inflammatory mediators and activated glial cells are prominent features of the inflammatory response in the brain of AD and contribute to neuronal cell death. Cox-2 is associated with Aß and is colocalized with Aß deposition in AD brain. In this study, we found that the intensity of Cox-2 bands and immunoreactivity in the CA region of the hippocampus and the dentate gyrus were preferentially elevated and colocalized precisely with Aß-42, suggesting that Cox-2 regulation may involve mechanisms in pathology of AD and inflammatory processes during AD pathogenesis. Expression of Cox-2 may have beneficial effects in the treatment of Tg mice with nonsteroid anti-inflammatory drugs (NSAIDs) because its expression in AD patients is influenced by treatment with NSAIDs. Thus, trials using NSE-hPS2w and NSE-hPS2m-Tg mice may be particularly promising.
NSE-hPS2w and NSE-hPS2m transgenic mice may closely mimic many neuropathological features of AD patients. Although these mice do not manifest complete AD pathology, they do appear to be good models of amyloid deposition, behavior defects, caspase-3 inhibition, and Cox-2 mediator, and thus could be useful for developing new therapeutic treatments for targeting various phenotypes showed in these Tg mice.
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ACKNOWLEDGMENTS |
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Received for publication September 4, 2001.
Revision received January 8, 2002.
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10 µg of tail DNA from Tg mice was subjected to Southern blot hybridization. Genomic DNA digested with EcoRI was probed with a 32P-labeled PS2 fragment to detect the integrated transgene. Fragment sizes, as calculated by the relative position of fragment of know size in 5.2, 6.2, 7.1 kb (lamda DNA digested with HindIII/HaeIII), are indicated at the right of the blot. Specific hybridization signals were visualized by autoradiography for 4 days at -70°C.
