APP intracellular domain is increased and soluble Aβ is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease
Introduction
A central hypothesis to the Alzheimer disease (AD) pathogenesis is that the amyloid-β peptide (Aβ) and/or Aβ-containing plaques play an important role in the development of the disease. Mutations in either the amyloid protein precursor (APP), presenilin 1 (PS1), or presenilin 2 (PS2) are linked to the inherited forms of the disease and result in an increased production of Aβ, particularly of the Aβ42 isoform (reviewed by Hardy, 1997). Aβ is derived from the proteolytic processing of the amyloid precursor protein Glenner and Wong, 1984, Kang et al., 1987, Masters et al., 1985 by the sequential action of β- and γ-secretases. β-Secretase [(or beta-site APP-cleaving enzyme (BACE)], a novel transmembrane aspartyl protease, cleaves APP at the Met595–Asp596 bond (APP695 numbering) to release the N-terminus of the Aβ peptides Hussain et al., 1999, Sinha et al., 1999, Vassar et al., 1999, producing a soluble sAPPβ fragment, and a membrane-bound C-terminal 99-amino acid fragment [β-C-terminal fragment (βCTF) or C99]. γ-Secretase activity that is contained within a complex including presenilin 1, nicastrin, PEN-2, and APH-1 (Francis et al., 2002) further processes the βCTF fragment. The exact position at which γ-secretase cleaves the C-terminal APP fragment is critical to AD pathogenesis. γ-Secretase can cleave the transmembrane domain of βCTF at alternative sites, to generate Aβ peptides of various lengths, with Aβ40 being the major species, followed by Aβ42. The longer Aβ42 species, which is more prone to aggregation, accounts for approximately 10% of Aβ (Vassar and Citron, 2000). The C-terminal product from this γ-cleavage, γCTF of 57–59 animo acids, has never been identified. However, a C-terminal fragment resulting from cleavage at Leu49–Val50 of the Aβ sequence (ε-cleavage) was identified and termed εCTF or APP intracellular domain (AICD) Gu et al., 2001, Sastre et al., 2001, Weidemann et al., 2002, Yu et al., 2001. AICD contains several internalization and trafficking motifs and has a potential transcriptional activity that resembles the NICD of Notch Baek et al., 2002, Cao and Südhof, 2001, Gao and Pimplikar, 2001. Another APP processing pathway that precludes Aβ production is cleavage of the ectodomain by α-secretase in the middle of the Aβ sequence, between Lys16 and Leu17, which produces a soluble APPα fragment, and a membrane-bound C-terminal fragment of 83 residues (αCTF or C83). α-Secretase belongs to the family of disintegrin metalloproteinases, such as tumor necrosis factor-α converting enzyme (TACE) and ADAM 10 (Hooper and Turner, 2002). Most mutations in APP, PS1, or PS2 that are associated with familial AD alter the γ-cleavage, resulting in increased levels of Aβ42 species and the corresponding CTFs Evin et al., 2002, Sastre et al., 2001, Sato et al., 2003, Selkoe, 1998.
The factors that influence Aβ metabolism and accumulation in sporadic AD have not yet been fully determined. Aging is the major risk factor for AD. The ε4 allele of ApoE (apolipoprotein E) is another prevalent risk factor for AD (Tanzi and Bertram, 2001) and plays an important role in Aβ deposition (Bales et al., 1997). ApoE is one of the major apolipoproteins in plasma and the principal cholesterol carrier in the brain. Increased cholesterol levels in serum and brain have been cited as a possible risk factor and correlated with an increased susceptibility to AD Notkola et al., 1998, Sparks, 1997. Interestingly, an epidemiological study illustrates that AD is more prevalent in countries with high dietary fat intake (Kalmijn et al., 1997). Studies of animal models have also shed further insight into the relationship between onset of AD and cholesterol levels. Young double transgenic APPsw and PS1M146V mice (by crossing the Tg2576 with the TgPS1 mice) fed on high-cholesterol diet showed accelerated Aβ deposition and increased βCTF but decreased sAPP in the brain (Refolo et al., 2000). On the other hand, an APP gene-targeted mouse model containing a humanized Aβ domain with Swedish (KM to NL) mutation showed reduced levels of Aβ and sAPP when fed a high-cholesterol diet (Howland et al., 1998).
It has been suggested in previous retrospective epidemiologic studies that there is a lower prevalence of probable AD cases in patients who received statins (HMG-CoA reductase inhibitors) to treat hypercholesterolemia, compared to patients who received no treatment Jick et al., 2000, Wolozin et al., 2000. However, more recent data obtained after a follow-up period of 3–5 years have shown that cholesterol lowering with simvastatin and pravastatin had no effect on cognition or onset of dementia Group, Heart Protection Study Collaborative Group, 2002, Shepherd et al., 2002. Treatment with statins greatly reduced cholesterol metabolite 24S-hydroxycholesterol in plasma (Locatelli et al., 2002) and in CSF (Simons et al., 2002) and caused a significant reduction in Aβ40 levels in mild AD subjects (Simons et al., 2002). However, in a case-controlled study of elderly nondemented subjects, the CSF level of Aβ42 was not associated with altered cholesterol metabolism (Fassbender et al., 2002). Tested in wild-type guinea pigs (Fassbender et al., 2001) or in a transgenic mouse model expressing APPsw or PS1M146V (Refolo et al., 2001), statins decreased Aβ production. The linkage between reduced cholesterol and Aβ production is not clear, because the secondary effects of statins, which include antioxidant and anti-inflammatory properties, may have an indirect influence on their effect on Aβ production (Cucchiara and Kasner, 2001).
There have been few studies so far of how increased dietary cholesterol may affect the development of AD. Since current literature shows divergent reports on the effect of dietary cholesterol on Aβ production, our study aimed to further investigate the effect of cholesterol on the production of Aβ and APP derivatives (in particular AICD) and to monitor its effect on general health and diet-related behavior in Tg2576 transgenic mice. The Tg2576 mouse model expresses human APP695 carrying the Swedish double mutation at codons 595 and 596 (APP695 numbering) under the hamster prion protein (PrP) promoter (Hsiao et al., 1996). The mice were placed on a high-cholesterol diet for 6 weeks. The results showed that hypercholesterolemia reduced the levels of Aβ but increased AICD in this mouse model.
Section snippets
Animals and diet conditions
All experimentation involving the use of mice was carried out under animal ethics approval. Hemizygous 12-month-old female Tg2576 mice, expressing the human APP gene containing the double Swedish (APPsw) mutation (Hsiao et al., 1996), and their nontransgenic (SJL/BL6) littermates were used, and the mice were genotyped by tail PCR analysis at 4 weeks of age. The mice were randomly selected for the experimental (n = 7) or control diet (n = 8). Nontransgenic (NTg) littermates were also placed on
Cholesterol levels are increased in the circulation, liver, and brain of mice fed a high-cholesterol diet
Twelve-month-old Tg2576 mice were placed on either the experimental high-cholesterol, high-fat diet, or the control basal diet used in previous studies Howland et al., 1998, Refolo et al., 2000 for 6 weeks. The average weights of the cholesterol-fed mice were not significantly different from the control diet-fed mice at the end of the experiment (6 weeks). The high-cholesterol diet also did not affect the general activity and behavior of the mice based on a battery of sensorimotor tests (data
Discussion
Clinical, epidemiologic, and biochemical studies suggest a link between cholesterol, APP processing, Aβ accumulation, and AD Jarvik et al., 1995, Kalmijn et al., 1997, Notkola et al., 1998, Papassotiropoulos et al., 2002, Simons et al., 2001, Sparks, 1997. The effect of dietary cholesterol on the expression and accumulation of Aβ in transgenic mouse models of AD has been controversial, with increases Refolo et al., 2000, Shie et al., 2002 and decreases (Howland et al., 1998) observed in
Acknowledgements
This work is supported in part by grants from the National Health and Medical Research Council of Australia and Prana Biotechnology Ltd. K.B. is supported by the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie. We thank Dr. Mark Murphy for advice on the open-field analysis, Drs. Karen Hsiao for the Tg2576 mice, Sam Gandy for the antibody 369, Ursula Mönning for the antibodies 13E9 and 6D5, Amanda Tammer and Aiqin Zhu for technical advice, and Ms. Tina
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