Conceived and designed the experiments: LP YML. Performed the experiments: LP LZ. Analyzed the data: LP. Contributed reagents/materials/analysis tools: LP LZ. Wrote the paper: LP YML.
The authors have declared that no competing interests exist.
Alzheimer disease (AD) is an age-related disorder. Aging and female gender are two important risk factors associated with sporadic AD. However, the mechanism by which aging and gender contribute to the pathogenesis of sporadic AD is unclear. It is well known that genetic mutations in γ-secretase result in rare forms of early onset AD due to the aberrant production of Aβ42 peptides, which are the major constituents of senile plaques. However, the effect of age and gender on γ-secretase has not been fully investigated. Here, using normal wild-type mice, we show mouse brain γ-secretase exhibits gender- and age-dependent activity. Both male and female mice exhibit increased Aβ42∶Aβ40 ratios in aged brain, which mimics the effect of familial mutations of Presenilin-1, Presenlin-2, and the amyloid precursor protein on Aβ production. Additionally, female mice exhibit much higher γ-secretase activity in aged brain compared to male mice. Furthermore, both male and female mice exhibit a steady decline in Notch1 γ-secretase activity with aging. Using a small molecule affinity probe we demonstrate that male mice have less active γ-secretase complexes than female mice, which may account for the gender-associated differences in activity in aged brain. These findings demonstrate that aging can affect γ-secretase activity and specificity, suggesting a role for γ-secretase in sporadic AD. Furthermore, the increased APP γ-secretase activity seen in aged females may contribute to the increased incidence of sporadic AD in women and the aggressive Aβ plaque pathology seen in female mouse models of AD. In addition, deceased Notch γ-secretase activity may also contribute to neurodegeneration. Therefore, this study implicates altered γ-secretase activity and specificity as a possible mechanism of sporadic AD during aging.
Alzheimer disease (AD) is a progressive and fatal neurodegenerative disease that is postulated to result from aggregation of toxic β-amyloid peptides (Aβ)
Overproduction and impaired clearance of Aβ can both contribute to increased plaque formation. Aβ peptides are produced by the sequential processing of the amyloid precursor protein (APP) by two proteases, β-secretase (BACE1) and γ-secretase
To determine the effect of gender and aging on
To determine if there was any age or gender influence on γ-secretase activity, we directly compared γ-secretase activity between male and female brains at varying ages. Total whole brain membrane fractions were isolated from male and female C57BL/6 mice at 1, 12, 18, and 24 months of age and
(A) Male mice exhibit reduced γ-secretase production of Aβ40 and Aβ42 peptides in aged brain compared to females but both males and females exhibit increased Aβ42∶Aβ40 ratios.
Although female mice had similar Aβ40 activity as males at 1 month, there was a transient 15–25% decline in Aβ40 activity in female mice at 12 and 18 months but by 24 months Aβ40 activity had returned to the 1 month baseline levels (
We next examined γ-secretase sensitivity to two structurally distinct inhibitors, L685,458 (L458) and Compound E. Aβ40
We then examined BACE1 activity in young (1 month) and aged (24 month) brain. Membrane isolated from mouse brain was incubated with an APP peptide substrate containing the Swedish mutation in the presence or absence of StatineV, a potent BACE1 inhibitor. Background was defined as the activity remaining in the presence of StatineV. At 1 month of age, female mice had about 15% more BACE1 activity than male mice (
Both male and female mice show an increase in BACE1 activity in aged brain. 10 µg brain membrane was assayed for BACE1 activity using a 12-mer FRET based substrate. Background was defined as activity remaining in the presence of 5–10 µM StatineVal. Values represent background subtracted fluorescent intensity (average±s.e.m., n = 14).
In addition to APP, γ-secretase is also known to process Notch1. Therefore, we wished to determine if there were any age or gender dependent changes in γ-secretase activity for Notch1 processing as we saw with APP processing. Total whole brain membrane fractions from 1, 12, 18, and 24 month old C57BL/6 mice were assayed for
Both male and female mice exhibit an age-dependent reduction in Notch1 γ-secretase activity.
Because there was a significant difference in γ-secretase activity between male and female brain at 24 months, we determined if there were any changes in the protein levels of the core subunits of γ-secretase which could account for the distinct sex specific activity profiles. Equal amounts of total membrane were resolved by SDS-PAGE and western blotted for the core γ-secretase subunits: nicastrin (Nct), presenilin (PS), Aph1aL, and Pen2. There was a reduction in Nct and PS1 levels at 24 months in male and female mice (
(A) Both female and male mice have a reduction in the protein levels of Nct and PS1, but not PS2, Aph1aL or Pen2, in aged brain. Equal amounts of total brain membrane was separated on SDS-PAGE and western blotted for the indicated subunits of γ-secretase. Representative blots shown. F = Female, M = Male. (B) There is a 50–60% reduction in total protein levels of Nct (left) and PS (middle and right) in 24 month male (open circles) and female mice (filled circles). Quantification of the relative band intensities of multiple western blots was calculated by measuring the area under the curve (AUC) using the gel analysis function of ImageJ software. Blots were internally normalized to the relative band intensity of 1 month female. Values represent percentage of 1 month (%1 mo) female AUC. (average±s.e.m. n = 4).
We have previously shown that γ-secretase activity does not always correlate with total expressed subunit levels
(A) Aged male mice have reduced photolabeling of PS1 compared to female mice at 24 months. The structure of compound 5 is indicated. The biotin moiety is marked with a dashed box, the photoreactive benzophenone is marked with a dashed circle, and the linear distance between the biotin moiety and the L458 backbone is 57Å as determined in ChemDraw. Equal amounts of total membrane were covalently photolabeled with compound 5 in the presence (L458, 1 month female shown as representative) or absence (DMSO) of non-biotinylated L458. Following denaturation with RIPA buffer, the labeled proteins were captured with streptavidin agarose, separated by SDS-PAGE and western blotted for PS1-NTF (upper panel) and PS1-CTF (lower panel). Representative blot shown. F = Female. M = Male. (B) Male mice have a 60% reduction in PS photolabeling at 24 months compared to females. Quantification of the relative band intensities of multiple photolabeling experiments was calculated by measuring the area under the curve (AUC) using the gel analysis function of ImageJ software. Blots were internally normalized to the relative band intensity of 1 month female. Values represent percentage of 1 month (%1 mo) female AUC. (average±s.e.m., n = 4).
Aging and gender are risk factors that are clearly associated with sporadic AD. However, whether or not these risk factors directly affect γ-secretase production of Aβ is not known. Multiple mouse models have been used to recapitulate Aβ pathology and have proved to be useful tools in understanding the pathogenesis of AD
Pathologic Aβ production can be mediated by (a) altered γ-secretase specificity resulting in increased Aβ42∶Aβ40 ratios leading to increased pathogenic plaque deposition (b) increased BACE1 activity which increases the levels of available APP substrate and therefore production of Aβ peptides (c) increased APP γ-secretase activity which results in increased Aβ production. In addition to pathogenic Aβ pathways, loss of Notch1 signaling due to decreased Notch1 γ-secretase activity may also lead to a neurodegenerative state (d). The data presented here implicates aging in pathways (a), (b), and (d) in the development of AD whereas (c) is an additional causative pathway implicated in the pathogenesis of neurodegeneration in females.
Photolabeling of PS1 suggests that the reduction in γ-secretase activity seen in aged males is due to either the reduced formation of active complexes or alterations to the active site architecture which reduce APP processing. However, the precise mechanism by which high levels of γ-secretase activity are maintained in aged female mouse brain remains unknown.
The decline in estrogen during menopause has been suggested to be a causative factor in the increased prevalence of AD in women. Early reports of hormone replacement therapy suggested a protective role of estrogen in the pathogenesis of AD
Interestingly, the gender dependant effects on γ-secretase activity appear to be specific for APP processing as both male and female mice exhibited a decline in Notch1 γ-secretase activity during aging and exhibit no gender associated differences at 24 months. Aged brain γ-secretase complexes, therefore, have gender dependent substrate processing abilities with female aged brain strongly favoring APP over Notch1 as a substrate and male brain showing no significant age related changes in its relative substrate preference. These data suggest that discrete γ-secretase complexes exist in brain which exhibit unique activity profiles, substrate processing abilities and regulation.
Notch signaling pathways are critical for cell fate decisions during development and direct proliferative, differentiation, and apoptotic responses
In summary, our data implicate a role for altered γ-secretase activity during aging in the development of sporadic AD and neurodegeneration; aged mice exhibit altered specificity for Aβ40 and Aβ42 production and decreased Notch1 processing, both of which are reminiscent of certain PS FAD mutations that result in the development of early onset AD. Furthermore, our demonstration that female mice have much greater γ-secretase activity in aged brain compared to males offers an additional model to explain the increased prevalence of AD in women and aggressive plaque pathology seen in female mouse models. Increased γ-secretase activity, along with altered Aβ42 and Aβ40 specificity, in aged brain, would potentially exacerbate the plaque load in females. This model would work in concert with previously demonstrated increases in BACE1 activity and reductions in neprilysin activity in female mouse models of AD. Additionally, loss of Notch1 γ-secretase activity in aged mouse brain may also augment the pathological consequences of increased Aβ42∶Aβ40 ratios and increased Aβ production by increasing neurodegeneration due to the loss of activated Notch1 mediated CRE-dependent gene expression. Therefore, our data help to clarify the roles of aging and female gender as risk factors for sporadic AD and advance our understanding of aberrant γ-secretase activity as a potential mechanism underlying the pathogenesis of sporadic AD.
Synthesis of L685,458 and Compound E were previously described
C57BL/6 mouse whole brain (male and female at 1, 12, 18, and 24 months of age) was obtained flash frozen from the NIA/NIH aged rodent tissue bank (Bethesda, MD) or special ordered from Taconic (Hudson, NY). Animals were tumor free with no gross pathologies or illnesses. Total membrane was isolated from two brains for each age and gender (
γ-Secretase activity was measured using the previously described electrochemiluminescence method
BACE1 activity was measured using a 12-mer FRET based substrate (TAMRA-Glu-Glu-Ile-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-QSY 7 amide). 10 µg membrane was incubated in BACE1 Assay Buffer (500 mM Sodium Acetate pH 4.5, 150 mM NaCl, and 0.1 mg/mL BSA) with 0.2% CHAPS (v/v) and 5 µM substrate in the presence or absence of 5–10 µM StatineV for 2 h at 37°C in the dark. BACE1 cleavage of the substrate was detected by excitation of the fluorophore at 530 nm and emission was monitored at 580 nm using a 50/50 beam splitting mirror (Envision, PerkinElmer). Activity is expressed as fluorescent intensity at 580 nm. Activity was measured in two independent membrane preparations (n = 4 animals total). Data points from two independent assays were averaged. P values were determined using a student t test in SigmaPlot8.0.
For western blotting, 20 µg total membrane was prepared with 4× reducing Lammeli sample buffer, separated by SDS-PAGE and transferred to PVDF membrane. Following blocking, the following antibodies were used for western blotting: Nct (1∶1000), PS1-NTF (1∶1000), PS1-CTF (1∶1000), PS2-CTF (1∶1000), Aph1aL (1∶250), and Pen2 (1∶500). Anti-mouse or anti-rabbit horseradish peroxidase conjugated secondary antibodies were used in concert with standard enhanced chemiluminescence detection methods. For photolabeling, 800 µg total membrane was pre-incubated with 2 µM non-biotinylated L458 or DMSO for 0.5 h at 37°C in Buffer B supplemented with 0.25% CHAPSO (v/v). Following pre-incubation, 20 nM compound 5 was added for 1 h at 37°C. Covalent crosslinking was performed by photoactivation of the benzophenone moiety at 350 nm for 0.75 h on ice. The reaction was denatured with 1× RIPA (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS (w/v), 1% Nonidet P40 (v/v), and 0.5% deoxycholic acid (w/v)) for 1 h at room temperature. The labeled complexes were captured with streptavidin agarose for 16 h at 4°C. The captured complexes were washed with 1× RIPA, eluted with 2× reducing Lammeli sample buffer, and resolved by SDS-PAGE. Western blotting was performed as described above. In all cases, blots are representative of a minimum of three independent experiments.
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We thank C. Chad Shelton for critical reading of the manuscript.