APP intracellular domain is increased and soluble Aβ is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease

https://doi.org/10.1016/j.nbd.2004.01.009Get rights and content

Abstract

Cholesterol is one of multiple factors, other than familial genetic mutations, that can influence amyloid-β peptide (Aβ) metabolism and accumulation in Alzheimer disease (AD). The effect of a high-cholesterol diet on amyloid precursor protein (APP) processing in brain has not been thoroughly studied. This study was designed to further investigate the role of cholesterol in the production of Aβ and APP intracellular domain (AICD) in 12-month-old Tg2576 transgenic mice. The mice were maintained on a high-cholesterol diet for 6 weeks. We found that diet-induced hypercholesterolemia increased the APP cytosolic fragment AICD and reduced sAPPα in the Tg2576 mice compared to the mice on a control basal diet. In addition, the levels of detergent-extracted Aβ40 were reduced, although no change in guanidine-extracted Aβ levels was observed. Full-length APP, α/βC-terminal fragment (α/βCTF), and β-secretase (BACE) were not different in the cholesterol-fed mice compared to the control diet-fed mice. This study suggests that a high dietary cholesterol in aged mice may not only influence Aβ metabolism, but also regulate the AICD levels. AICD has a proposed role in signal transduction and apoptosis, hence modulation of AICD production could be an alternative mechanism by which cholesterol contributes to AD pathogenesis.

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

References (69)

  • J. Hardy

    Amyloid, the presenilins and Alzheimer's disease

    Trends Neurosci.

    (1997)
  • D.S. Howland et al.

    Modulation of secreted beta-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol

    J. Biol. Chem.

    (1998)
  • I. Hussain et al.

    Identification of a novel aspartic protease (Asp 2) as beta-secretase

    Mol. Cell. Neurosci.

    (1999)
  • N. Ida et al.

    Analysis of heterogeneous βA4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay

    J. Biol. Chem.

    (1996)
  • H. Jick et al.

    Statins and the risk of dementia

    Lancet

    (2000)
  • W.T. Kimberly et al.

    The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates to the nucleus in a Notch-like manner

    J. Biol. Chem.

    (2001)
  • M.P. Mattson et al.

    Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β-amyloid precursor protein

    Neuron

    (1993)
  • A. Papassotiropoulos et al.

    24S-hydroxycholesterol in cerebrospinal fluid is elevated in early stages of dementia

    J. Psychiatr. Res.

    (2002)
  • I.H. Park et al.

    Lovastatin enhances Abeta production and senile plaque deposition in female Tg2576 mice

    Neurobiol. Aging

    (2003)
  • L.M. Refolo et al.

    Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model

    Neurobiol. Dis.

    (2000)
  • L.M. Refolo et al.

    A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease

    Neurobiol. Dis.

    (2001)
  • T. Sato et al.

    Potential link between amyloid beta-protein 42 and C-terminal fragment gamma 49–99 of beta-amyloid precursor protein

    J. Biol. Chem.

    (2003)
  • D.J. Selkoe

    The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease

    Trends Cell Biol.

    (1998)
  • J. Shepherd et al.

    Pravastatin in elderly individuals at risk of vascular disease (PROSPER): a randomised controlled trial

    Lancet

    (2002)
  • D.L. Sparks et al.

    Induction of Alzheimer-like β-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol

    Exp. Neurol.

    (1994)
  • R.E. Tanzi et al.

    New frontiers in Alzheimer's disease genetics

    Neuron

    (2001)
  • R. Vassar et al.

    Abeta-generating enzymes: recent advances in beta- and gamma-secretase research

    Neuron

    (2000)
  • S. Wahrle et al.

    Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains

    Neurobiol. Dis.

    (2002)
  • W.G. Wood et al.

    Brain membrane cholesterol domains, aging and amyloid beta-peptides

    Neurobiol. Aging

    (2002)
  • C. Yu et al.

    Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma-secretase processing of the APP and Notch1 transmembrane domains

    J. Biol. Chem.

    (2001)
  • K.R. Bales et al.

    Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition

    Nat. Genet.

    (1997)
  • M. Burns et al.

    Presenilin redistribution associated with aberrant cholesterol transport enhances beta-amyloid production in vivo

    J. Neurosci.

    (2003)
  • X.W. Cao et al.

    A transcriptively active complex of APP with Fe65 and histone acetyltransferase Tip60

    Science

    (2001)
  • G. Evin et al.

    Carboxyl-terminal fragments of the amyloid precursor protein in Alzheimer's disease brain: detection of γ-secretase products

    Brain Aging

    (2002)
  • Cited by (74)

    • Animal Models of Alzheimer's Disease

      2017, Animal Models for the Study of Human Disease: Second Edition
    • Pioglitazone improves the ability of learning and memory via activating ERK1/2 signaling pathway in the hippocampus of T2DM rats

      2017, Neuroscience Letters
      Citation Excerpt :

      The supernatant was determined using Bradford method to measure the total protein content. Western Blot analysis was performed according to previously described procedures [21] to measure the target proteins. Briefly, Mix the protein sample with the Laemmli sample buffer in 1:1 and boil for 5 minutes, then loaded onto 12% gradient gels and electrophoresed.

    • The Influences of Dietary Sugar and Related Metabolic Disorders on Cognitive Aging and Dementia

      2016, Molecular Basis of Nutrition and Aging: A Volume in the Molecular Nutrition Series
    View all citing articles on Scopus
    View full text