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Caloric restriction attenuates ß-amyloid neuropathology in a mouse model of Alzheimer’s disease

Jun Wang^*, Lap Ho^*, Weiping Qin^*, Anne B. Rocher, Ilana Seror^*, Nelson Humala^*, Kruti Maniar, Georgia Dolios^‘||, Rong Wang^‘||, Patrick R. Hof^,, and Giulio Maria Pasinetti^*,,,1

^* Neuroinflammation Research Laboratories, Department of Psychiatry,
Department of Neurosciences,
Department of Geriatrics and Adult Development,
Advanced Imaging Program, and
^‘|| Department of Human Genetics, Mount Sinai School of Medicine, New York, New York, USA

¹Correspondence: Mount Sinai School of Medicine, Neuroinflammation Research Laboratories, Department of Psychiatry, Box 1230, One Gustave L. Levy Pl., New York, NY 10029, USA. E-mail: giulio.pasinetti{at}mssm.edu

SPECIFIC AIMS

Recent prospective studies indicate that increased caloric intake is a risk factor for Alzheimer’s disease (AD). Though there is evidence supporting a potential neuroprotective role of caloric restriction (CR) in the brain, there is no information whether a CR regimen may influence AD. A fundamental problem in AD is the aberrant generation of amyloidogenic ß-amyloid peptides in the brain leading to abnormal deposition of neuritic plaques, which is a neuropathological landmark in AD. Based on this consideration, in this study using a mouse model of AD-type amyloidosis we tested the hypothesis that CR may beneficially influence AD through mechanism that prevent Aß generation and eventually neuritic plaque deposition in the brain.

PRINCIPAL FINDINGS

1. Chronic treatment of Tg2576 mice with a caloric restriction dietary regimen resulted in significantly lower AD amyloid neuropathology in the brain
To test the hypothesis that CR can modulate amyloidosis, 3-month-old Tg2576 mice, which develop AD amyloid neuropathology by 8–10 months of age, were maintained for 9 months on a low-carbohydrate CR regimen resulting in a 30% lower daily caloric intake compared with that consumed by age- and gender-matched control Tg2576 mice fed ad libitum (AL). When Tg2576 mice were examined for AD-type neuropathology at 12 months of age, we found that 9-month CR treatment almost completely prevented cortical and hippocampal AD-type amyloid plaque development (Fig. 1 A, B) relative to animals in the AL-fed group. Consistent with this evidence, we noted commensurately lower concentrations of amyloidogenic Aß_1-40 and Aß_1-42 peptides in the neocortex and hippocampus as evaluated by ELISA assay, relative to AL-fed controls (Fig. 1C) . No detectable change in total full-length amyloid precursor protein (APP) level was noted in either brain region of CR vs. AL-fed Tg2576 mice (not shown).

View larger version (32K): [in this window] [in a new window]   Figure 1. CR attenuates AD-type amyloid neuropathology in brain of 12-month-old Tg2576 mice. A) Stereological assessment of neocortical and hippocampal Aß-amyloid plaque burden (thioflavin-S positive volume as % of regional volume). B) Representative thioflavin-S positive Aß amyloid plaque neuropathology in the neocortex (CTX) and hippocampal subicular (S) region in AL (left) and CR mice (right). C) Assessment of neocortical and hippocampal Aß1-40 and Aß1-42 peptide concentrations in CR and AL-fed control Tg2576 mice. D, E) IP-MS spectra of neocortical Aß peptide profiles of CR and AL-fed control mice after immunoprecipitation with 4G8 (D) and 6E10 (E) antibodies. The Aß IP-MS spectra from 4G8 IP was normalized to internal standard Aß12-28; Aß IP-MS spectra from 6E10 was plotted using a relative scale to the peak intensity of Aß1-40 in the AL-fed control group and referenced to their relationship in the 4G8 IP to compare Aß fragment peptides. D, E) MS peaks corresponding to Aß peptides are indicated with Aß peptide sequence number. Peaks labeled 1–402+ and insulin2+ represent doubly protonated Aß1-40 peptide and insulin molecular ions, respectively. Aß12-28 was added during the IP procedure and used as internal standard (int. std.) ions. Bar graphs: group mean ± SE, n = 5–7 animals/group; *P < 0.01, **P < 0.005, 2-tailed t test, CR vs. AL groups. B) Arrowheads point to thioflavin-S positive Aß amyloid plaques; length bar = 30 µM.

2. The anti-amyloidogenic role of CR is associated with selective promotion of anti-amylogenic -secretase activity
We explored APP processing and Aß peptide generation using immunoprecipitation (IP) mass spectrometry (IP-MS). Using 4G8 antibody for Aß IP, we found a relative proportional reduction in Aß_1-37, Aß_1-38, and Aß_1-39 peptide content in the neocortex of the CR group compared with the AL-fed control group (Fig. 1D ). This evidence, together with our observation that the concentration of the 7 kDa carboxyl-terminal fragment (CTF)- cleavage product of APP (an index of -secretase activity) was unchanged in the neocortex of the CR group relative to AL-fed controls, is consistent with the possibility that -secretase activity was not involved in the CR-associated anti-amyloidogenic activity. However, in additional Aß IP-MS studies using 6E10 antibody, we found a major elevation in Aß_1-16 peptide fragment concentration in the neocortex of the CR group that was not detected in the AL-fed controls (Fig. 1E ). Because -secretase can cleave APP, eventually resulting in the generation of Aß C-termini fragments ending at the AA residue leucine16 of Aß, we continued to explore the role of CR in -secretase activity in the brain.

Cleavage of APP by -secretase releases the amino-terminal extracellular domain known as soluble -secretase amyloid precursor protein (sAPP) domain coincidental with elevation in membrane-bound -secretase-cleaved APP carboxyl-terminal fragment (CTF)-. We found that CR in Tg2576 mice resulted in a >2-fold elevation in concentration of neocortical sAPP (Fig. 2 A) and membrane-associated CTF- (Fig. 2B ), relative to AL-fed control Tg2576 mice. The 1.6-fold increase in CTF- was somewhat less relative to sAPP, presumably because of further cleavage of CTF- by -secretase. The abundance of CTF-ß signal was at the limit of detection in the neocortex of both CR and AL-fed Tg2576 mice, preventing reliable quantification (Fig. 2B ).

View larger version (27K): [in this window] [in a new window]   Figure 2. CR promotes -secretase activity in the brain of Tg2576 mice relative to AL-fed control mice. A) Assessment of changes in neocortical sAPP concentration expressed as % of full-length APP immunoreactivity; inset: representative sAPP and full-length APP immunoreactive signal in the same sample. B) Assessment of APP CTF- cleavage product concentrations; inset: representative CTF- (and CTF-ß) and full-length APP immunoreactive signal from the same immunoblot. C) Assessment of mature and proform ADAM10 concentrations in the same neocortical specimen relative to ß-actin immunoreactive signal; inset: representative immunoblot of proform and mature forms of ADAM10 protein species and ß-actin immunoreactivities. D) Fluorimetric assessment of -secretase activity. Bar graphs represent group mean ± SE, n = 5–7 mice/group; *P < 0.02, **P < 0.01, 2-tailed t test, AL vs. CR group.

3. CR may influence -secretase activity in the brain in part by selectively promoting the generation of mature, catalytically active ADAM10 species
Recent evidence indicate that the proteinase ADAM10 (a disintegrin and metalloproteinase) may act as an -secretase. The 62 kDa mature ADAM10 protein species is known to act as an -secretase in vitro and to cleave Aß-derived peptides at leucine₁₆. We found that the CR diet regimen resulted in a 30% elevation of neocortical mature ADAM10 species concentration (Fig. 2C ), coinciding with a commensurate elevation in neocortical -secretase activity (assessed fluorimetrically), compared with AL-fed control mice (Fig. 2D ). No detectable change in proform ADAM10 species concentration was noted in the neocortex of the CR group relative to the AL-fed control group (Fig. 2C ). In parallel studies, we found that the CR regimen in Tg2576 mice did not change the concentration of the proform or mature form of either the ADAM17 or ADAM9 species, relative to AL-fed control Tg2576 mice (not shown). Similarly, there was no detectable change in the neocortical concentration of the BACE1 species or of ß-secretase activity in response to CR relative to the AL-fed control group (not shown).

CONCLUSIONS AND SIGNIFICANCE

Our studies support the hypothesis that CR may prevent AD-type amyloid neuropathology through mechanisms that influence -secretase activity in the brain, possibly by promoting the generation of mature, catalytically active ADAM10 species (Fig. 3 ). Since -secretase proteolysis of the APP sequence within the Aß peptide would preclude the generation of amyloidogenic Aß peptides, our studies suggest that CR may provide an attractive anti-amyloidogenic strategy by promoting -secretase activity in the brain. Because AD-type amyloid neuropathology is undetectable in the brain of 3-month-old Tg2576 mice (the age when our mice were initially exposed to the CR dietary regimen) our studies support the possibility that CR, by promoting -secretase activity preventively, (possibly through mechanisms involving ADAM10 maturation) may result in reduced AD-type amyloid neuropathology. This possibility is supported by recent evidence showing that decreased -secretase activity in the brain of dominant negative ADAM10 - APP_(v717) double transgenic mice develop more amyloid neuropathology than controls.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram. CR may prevent AD-type amyloid neuropathology through mechanisms that influence -secretase activity in the brain, possibly by promoting the generation of mature, catalytically active ADAM10 species.

Current strategies to treat AD are aimed at preventing formation of amyloidogenic Aß peptides by modulating - and ß-secretase activities necessary for generation of Aß peptides. It has, however, been difficult to find safe, selective ß- and -secretase inhibitors, mainly because of the influence of these inhibitors on other cellular substrates. Thus, our evidence showing that CR may positively influence -secretase the brain might prove in the future the basis of potential novel preventative measure aimed at delaying the onset of AD neuropathology. We cannot, however, exclude the possibility that CR might also influence other mechanisms, eventually resulting in decreased amyloid deposition in the brain by promoting -site cleavage of APP by other proteases (e.g., plasmin) or degradation of released Aß. Since -secretase cleavage of APP releases sAPP, which is well known for its neuroprotective properties, our study tentatively suggests that promoting CR dietary regimen may also result in increased brain repair activities as a consequence of sAPP neurotrophic function.

Consistent with the evidence that caloric intake may be a risk factor for AD, this study for the first time offers a rational basis for a potential future preventive measure aimed at delaying the onset of AD amyloid neuropathology via control of dietary intake.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3182fje;

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