Aberrant expressions of pathogenic phenotype in Alzheimer's diseased transgenic mice carrying NSE-controlled APPsw
Introduction
Alzheimer's disease (AD), the most common cause of dementia in elderly humans, occurs when neurons in the memory and cognition regions of the brain are accompanied by massive accumulation of abnormal fibrous amyloid β-proteins (Aβ). Aβ are deposited as extracellular senile plaques composed of the 39–43 amino-acid-long peptide, derived from the β-amyloid precursor protein (APP), caused by cleavage with β-secretase and γ-secretase. The α-secretase cleaves the middle of the Aβ region, releasing a secreted ectodomain that contains the first 16 amino acids of the Aβ (sAPPα). In contrast, β-secretase, the novel transmembrane asparatic protease, BACE (Vassar et al., 1999), cleaves between Met-671 and Asp-672, producing the amino terminal end of the Aβ and ending with Met-671 (sAPPβ). Further processing of the carboxyl-terminal end of sAPPβ, by the activity of γ-secretase, leads to the release of Aβ.
The APP gene contains 19 exons, three of which (exons 7, 8, and 15) are presented as alternative splicing, and have been found to be expressed in a variety of tissues, as a family of differentially spliced transcripts, ranging from 695 to 770 amino acids (Rockenstein et al., 1995). Three different APP isoforms were designated, according to the number of amino acids: (i) The APP751 contains 56 amino acid domains (encoded by exon 7), with homology to the Kunitz family of serine protease inhibitors (Ponte et al., 1988). (ii) The APP770 contains an additional 19-amino-acid domain (encoded by exon 8) with homology to the MRC OX-2 antigen, which is found on the surface of neurons and immune cells Golde et al., 1990, Jacobson et al., 1991, Mucke et al., 1994. (iii) The APP695 is a splicing variant lacking the Kunitz domain, which was preferentially expressed in neuronal tissues Neve et al., 1988, Tanzi et al., 1993.
A number of APP mutations, which increase the production of Aβ, are tightly associated with the development of AD by changing the amino acids close to either side of the peptide. Missense mutations of APP, occurring at codon 717 (London mutations), have been shown to produce increased amounts of the more amyloidogenic form of the 42 amino acids of the Aβ from the APP Cahoun et al., 1998, Irizarry et al., 1997a, Johnson-Wood et al., 1997, Suzuki et al., 1994. Another mutation, at the 670/671 codon in APP (APPsw), leads to enhanced cleavage by the β-secretase, and increased Aβ formation (Irizarry et al., 1997b). Thus, mutations in the genes for Alzheimer's disease influence the processing of the amyloid precursor protein (APP), causing elevated levels of the Aβ-42 deposit.
The Aβ-42 deposit leads to Alzheimer-like behavior phenotypes that are linked to neuronal cell death through potential signaling events. In particular, the mechanisms of the mitogen-activated protein kinase (MAPK) pathway that underlie the Aβ-42 deposition in AD are poorly understood. It has been demonstrated that exposure of cortical neurons to Aβ induces the activated c-Jun N-terminal kinase (JNK) required for the phosphorylation and activation of the c-Jun transcription factor (Troy et al., 2001). p38 is also a part of a signal transduction pathway activated in the cortex of the transgenic model mice (Savage et al., 2002). In addition, activation of the extracellular signal-regulated kinase (ERK) of the MAPK cascade in the hippocampus is required for memorial formation (Atkins et al., 1998), although ERK is not activated in the cortex of AD model mice (Savage et al., 2002). These results raised the possibility of three members (JNK, p38, and ERK) of the MAPK family, contributing, either directly or indirectly, to Aβ-dependent cell death, or brain function, in AD patients. Furthermore, tau hyperphosphorylation is thought to be a critical event in the pathogenesis of AD. In an AD brain, tau is abnormally hyperphosphorylated, and aggregates into paired helical filaments (PHF) Iqbal et al., 1989, Lee et al., 1991 that are accumulated in neuronal cell bodies as neurofibrillary tangles (NFT), a histopathological hallmark of AD (Arnold et al., 1991). At present, it is unknown whether the APP in transgenic mice affects the MAPK and tau hyperphosphorylation, or if these are required for the progression of AD.
On the other hand, the expression of neuronal Cox-2 is elevated in the AD brains Pasinetti and Aisen, 1998, Yasojima et al., 1999, and immunostaining for Cox-2 correlates with the number of amyloid plaques Ho et al., 1999, Yasuji et al., 2001. Cox-2 was preferentially localized in the neurofibrillary tangle positive neurons, with damaged axons (Yasojima et al., 1999). These findings suggest that neuronal Cox-2 may contribute to the pathology of Alzheimer's disease by promoting memory dysfunction and neuronal apoptosis, which are regulated by MAPK phosphorylation (Dean et al., 1999).
An increase in Aβ, due to APP mutation, leads to apoptosis in the brains of AD patients Kitamura et al., 1998, Perry et al., 1998. In particular, caspase-3 has been shown as significant in the development of the nervous system Kuida et al., 1996, Srinivasan et al., 1998, as well as in AD brains (Oka and Takashima, 1997). Thus, caspase-3 is thought to be responsible for the apoptotic signal, but its functional mechanism to AD remains unknown. Such information is important for understanding the molecular mechanism of neuronal cell death in AD.
In this study, transgenic mice overexpressing APPsw under the control of the NSE promoter were produced, and these were used to examine the behavioral deficits. We also examined whether this overexpression of the APPsw transgene was associated with several pathogenic phenotypes. Alterations in the behavior and Aβ-42 depositions were shown at 12 months of age in the transgenic mice. There were also increased activities of the JNK and p38 kinases, including tau phosphorylation, when the brains were examined by Western blot analysis. Furthermore, Cox-2 was highly expressed in the brains of the transgenic mice relative to the control mice. In parallel, it was found that the caspase-3 activation and TUNEL-stained nuclei were enhanced in the brains of transgenic mice.
Section snippets
Gene construction
A plasmid, pNSE-APPsw, containing APPsw under the control of NSE promoter, was constructed. The APPsw sequence was amplified by a polymerase chain reaction (PCR), with full-length APP695sw as a template. The CMVAPP695sw construct, containing APPsw695, was a gift from Dr. Tae-Wan Kim, from Columbia University. The primers used for the amplification were the APPsw sense primer, 5′-TCTAG ATCGC GATGC TGC-3′ (corresponding to nucleotide 143–154 of APPsw), and the APPsw antisense primer, 5′-GTCTA
Generation of transgenic mice
NSE-APPsw (Fig. 1A), with the prokaryotic sequence eliminated, was used for microinjection, and the founder mice were analyzed for the presence of the NSE-APP transgene by DNA-PCR analysis (Fig. 1B). From a total of nine offsprings (five male and four female), two transgenic mice, one female (#1228) and one male (#1223) carrying the NSE-APPsw, were identified. In the other line (Am1228), the Aβ-42 levels were higher than those in control mice, and behavioral deficits appeared at 12 months,
Discussion
Transgenic mice models are useful for understanding the fundamental mechanisms of the pathological changes underlying AD, and are needed to optimize therapeutic drugs. In this study, transgenic mice overexpressed APPsw under the control of the NSE promoter were produced, and showed evidence of behavioral deficit. In the water maze tests, 12-month-old transgenic mice had longer escape latencies, across the former platform location, than the age-matched control mice. These transgenic mice also
Acknowledgements
We would like to thank both Sun M. Choi, BS, and Mi K. Chang, BS, the animal technicians, for directing the animal facility at the Division of Laboratory Animal Resources. Our special thanks must be given to Dr. Tae-Wan Kim, from Colombia University, for his gift of the mutant the APPsw and for his valuable discussions. This research was supported by grants to Dr. Yong K. Kim from the Korean Ministry of Health and Welfare (01-PJ1-PG3-20500-0129) and the Korea FDA.
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