Transient pharmacologic lowering of Aβ production prior to deposition results in sustained reduction of amyloid plaque pathology
© Das et al.; licensee BioMed Central Ltd. 2012
Received: 22 June 2012
Accepted: 6 August 2012
Published: 14 August 2012
Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Disease modifying therapies targeting Aβ that are in development have been proposed to be more effective if treatment was initiated prior to significant accumulation of Aβ in the brain, but optimal timing of treatment initiation has not been clearly established in the clinic. We compared the efficacy of transient pharmacologic reduction of brain Aβ with a γ-secretase inhibitor (GSI ) for 1–3 months (M) treatment windows in APP Tg2576 mice and subsequent aging of the mice to either 15M or 18M.
These data show that reducing Aβ production in a 2-3M windows both initiated and discontinued before detectable Aβ deposition has the most significant impact on Aβ loads up to 11M after treatment discontinuation. In contrast, initiation of treatment for 3M windows from 7-10M or 12-15M shows progressively decreasing efficacy.
These data have major implications for clinical testing of therapeutics aimed at lowering Aβ production, indicating that; i) these therapies may have little efficacy unless tested as prophylactics or in the earliest preclinical stage of AD where there is no or minimal Aβ accumulation and ii) lowering Aβ production transiently during a critical pre-deposition window potentially provides long-lasting efficacy after discontinuation of the treatment.
Alzheimer’s disease (AD) is the leading cause of dementia among the elderly. Numerous studies in humans have demonstrated the sequential development of various pathological features that characterize AD and the relationship of these pathologies to diagnosis of dementia . These studies support the amyloid cascade hypothesis which posits that accumulation of Aβ aggregates triggers a series of downstream pathologies that results in clinical AD . AD has recently been proposed to have three pre-clinical stages, characterized in sequence by the development of Aβ pathology (stage 1), the presence of plaque pathology, neurofibrillary pathology and initial evidence for neurodegeneration (stage 2), and finally by the presence of stage 2, pathologies plus evidence for subtle cognitive decline (stage 3). This progression likely occurs over a 10–20 year timeline .
The amyloid cascade hypothesis predicts that attenuating or preventing Aβ aggregation and accumulation will attenuate or prevent the subsequent development of AD. Strong experimental support for the amyloid hypothesis has supported the rationale for many therapeutic approaches targeting Aβ. Major investments have been made to develop inhibitors or modulators of Aβ production . γ-secretase inhibitors (GSIs), β-secretase inhibitors (BSIs) and γ-secretase modulators (GSMs) have been or are currently being tested in human AD trials. Both the GSM tarenflurbil (R-flurbiporfren), and the GSI semagacestat, failed to show efficacy in phase III human trials [4, 5]. Several other GSMs, GSIs, and BSIs are currently in early stage trials. Based on the clinical failures of tarenflurbil and semagacestat as well as other anti-Aβ therapies not targeting Aβ production but its aggregation or clearance, we and others have begun to question whether targeting of Aβ in symptomatic AD may be a futile endeavor .
Results and discussion
In vitro and in vivodosing studies with Ly-411,575
Brain and plasma Aβ40 levels following in vivo GSI treatment in Tg2576 mice
Strain of Mice
Length of Treatment
% Aβ40 ReductionBrain
% Aβ40 ReductionPlasma
Oral (Suspension in Kool-Aid)
Oral (Formulated In Rodent Chow)
Transient Aβ reduction dramatically reduces the subsequent accumulation of amyloid plaques
Plasma Aβ40 levels following transient GSI treatment in Tg2576 mice
Plasma Aβ40 (pM)
341.3 ± 20.1 (81%)
1742.6 ± 103.5
327.5 ± 18.5 (79%)
1520.7 ± 100.8
427.3 ± 31.4 (70%)
1388.4 ± 223.2
Because Aβ deposition in mice follows an exponential course, we sought to determine how the observed reductions in Aβ accumulation correlate with Aβ accumulation to untreated mice. We compared Aβ levels in the brains of untreated Tg2576 mice from various ages ranging from 10–18 months and plotted the natural logarithm transformed value of these against Aβ levels from the 4-7M LY treated mice, aged to 15M and 18M (Figure 2D). To minimize variance, we assayed all of the samples in a single set of ELISA studies. This analysis showed that the 4 -7M GSI treatment time frame “shifts” amyloid depositing by approximately 3 months, essentially delaying Aβ accumulation by a time roughly equivalent to the treatment window.
We next attempted to determine whether this 4-7M narrow therapeutic “window” can be further refined. To do this, we transiently dosed Tg2576 mice with LY for 1, 2 or 3M intervals between the 4-7M age time frame and again aged the mice to 15M. Aβ levels were again significantly reduced in this second 4-7M LY cohort (~59% reduction) at 15M of age compared to controls (Figure 2E). Similarly, i) the 4-6M LY cohort showed a significant reduction in Aβ levels (~51% reduction) and; ii) the 4-5M LY group showed a modest but non-significant reduction in Aβ levels (~ 24% reduction) (Figure 2E). However, in the remaining 5-6M, 6-7M or 5-7M LY cohorts tested, none showed any significant reductions in Aβ levels at 15M compared to controls (Figure 2E). Since we show that the 4-7M LY “shifts” amyloid deposition equivalently to the duration of treatment, we wondered whether we will see a similar shift in amyloid deposition in the shorter treatment intervals examined that showed efficacy, e.g., in the 4-5M and 4-6M cohorts. Again, we compared Aβ levels in the brains of untreated Tg2576 mice from various ages ranging from 10–15 months against Aβ levels from the 4-5M, 4-6M, and 4-7M LY treated cohorts aged to 15M (Figure 2F). This analysis seems to show that the 4-5M GSI treatment “shifts” amyloid depositing by approximately 1 month, however, both the 4-6M and 4-7M treatment intervals essentially show a similar shift in Aβ accumulation, namely by roughly 3 months (Figure 2F). These data would suggest, at least within the 4-7M time frame that we have studied, that the 4-6M age interval may play a critical role in the early seeding phases of amyloid deposition. Additional studies, using either earlier treatment times (e.g. starting at 3M of age) or longer treatment intervals may be necessary to fully appreciate the usefulness of this early “therapeutic window” to achieve the best possible outcome on subsequent amyloid deposition.
Transient GSI does not permanently alter APP levels or processing
Our current data show that transiently lowering Aβ production in Tg2576 mice during the pre-deposition “seeding” phase has a major impact on subsequent Aβ accumulation with the effect persisting for at least 11M. Later treatment windows showed decreasing efficacy of treatment. These current data have major implications for trial design targeting Aβ production in humans. Indeed, they would suggest that efficacy in terms of plaque reduction is likely to be maximal during seeding phase, and rapidly decline if treatment is initiated during the exponential phase of deposition. A recent report  using passive immunotherapy provided more limited evidence that a similar effect may be observed with anti-Aβ passive immunotherapy initiated during the early deposition phase persisting for 3 months after discontinuation of treatment. Given the plethora of anti-Aβ therapies currently in development, it will be essential to determine for each modality whether efficacy is both maintained after discontinuation and similarly influenced by timing of the treatment.
The near equivalence of the delay in subsequent deposition to the length of the treatment window during a pre-deposition phase suggests that the GSI treatment from 4-7M alters or reverses seeding of Aβ. Indeed, there are no plaques forming during this period of time. Future studies will be necessary to determine the exact mechanisms by which lowering Aβ in this window alters subsequent deposition. As even in vitro detection of Aβ nucleation relies on indirect assays that have limited sensitivities, these studies will likely require a better understanding of nucleation and better assays to detect it. Developing such assays will be critical as direct translation of these studies would be facilitated by defining the equivalent window in humans. Regardless of mechanism, these preclinical window therapy studies have major implications regarding the potential of prophylactic therapy targeting Aβ production in humans. Not only do these data suggest that prophylactic therapy in the appropriate window will be more efficacious then initiating therapy in individuals with any preexisting Aβ pathology, but they suggest that such therapy, even if discontinued might delay subsequent onset AD by a time roughly equivalent to the time of treatment.
APP Tg2576 mice were generated, maintained and genotyped as described previously . All animal husbandry procedures performed were approved by the Mayo Clinic Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines. All animals were housed three to five to a cage and maintained on ad libitum food and water with a 12 h light/dark cycle.
LY-411, 575 (LY) was synthesized at the Mayo Clinic Chemical Core as previously described . LY at a concentration of 16.75 mg per kg of diet was homogenously incorporated into Harlan Teklad 7012 kibble chow by Research Diets, Inc, New Jersey). Based upon dietary consumption at this age, these diets were designed to deliver 2.5 mg of LY on average per day. Tg2575 mice (n = 7-15/group/experiment) were transiently treated with LY chow at 1, 2 or 3 months interval (between 4–7 months of age), from 7–10 months of age and from 12–15 months of age. Untreated control mice from each age group were as aged and used for analysis. Groups of mice (n =3) were sacrificed immediately after the end of each treatment time-point to access efficacy of LY inhibition. After treatment was halted, mice were then aged till 15 months or 18 months of age and then sacrificed for Aβ analysis. Chow consumption, general health and body weight were monitored on a weekly basis in all treatment groups.
Biochemical Aβ ELISA assay
Snap-frozen hemi-forebrains from Tg2576 mice were two-step sequentially extracted in 2% SDS buffer followed by 70% formic acid (FA) as described previously . Aβ levels from brain lysates or plasma were the measured using sandwich ELISA techniques as described previously  with end specific mAbs 2.1.3 (human Aβx-42 specific, Mayo) and mAb 13.1.1 (human Aβx-40 specific, Mayo) for capture and HRP-conjugated mAb Ab5 (human Aβ1–16 specific, Mayo) for detection.
Sagittal sections of paraffin embedded sections were used for analysis. Immunohistochemical staining was done using pan-Aβ antibody (mAb 33.1.1; Mayo), mAb 13.1.1 (human Aβx-40 specific, binds cored plaques and CAA; Mayo) as previously described . Immunohistochemically stained sections were captured using the Scanscope XT image scanner (Aperio, Vista, CA, USA) and analyzed using the ImageScope program. Aβ plaque burden was then calculated using the Positive Pixel Count program (Aperio). At least 5 sections/brain 30 μm apart, were used and averaged by a blinded observer to calculate plaque burden.
2% SDS solubilized brain lysate samples were separated on Bis-Tris 12% XT gels (Bio-Rad, Hercules, CA, USA) and probed with the antibody CT20 (anti-APP C-terminal 20 amino acid; 1:1000; P.D) and anti-actin (1:1000; Sigma, St. Louis, MO, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Cell isolation, in vitropolarization and Immuno-phenotyping
Single cell suspensions were prepared aseptically from spleens from control or GSI -treated mice. CD4+ T cells were positively selected using the BD-IMAG system and CD4 + DM particles (BD Biosciences, San Diego, CA), according to the manufacturer’s directions. Twelve-well tissue culture plates were precoated with anti-hamster Ig (Sigma), then with antibodies specific for CD3ε and CD28. Equal numbers of cells (3x106) were added to each well. For TH1 polarizing conditions, anti-IL4 (10μg/ml) plus IL-12 (1 ng/ml) was added to culture wells; for TH2 polarizing conditions, anti-IFNγ (10μg/ml) plus IL-4 (1 ng/ml) was added to culture wells. No additional reagents were added to cells cultured under neutral conditions. After 24 hours, all wells were pulsed with IL-2 (10 ng/ml). After 96 hours, supernatants were collected and frozen at -80C until analyzed by ELISA for cytokine secretion. IFNγ and IL-4 cytokine secretion was measured using ELISA kits per manufactures instructions (BD Pharmingen). For cell surface staining, approximately 1x106 cells (each antibody) were stained with the following antibodies individually: IgD-FITC, biotinylated anti-CD19 plus strep-PE, CD4-FITC, CD8-PE (BD Pharmingen). Data were analyzed using an LSRII flow cytometer (BD Biosciences) and FACSDiva software for acquisition and analysis.
All statistics were done using Graphpad Prism (Version 5.0). Comparisons of multiple groups were done by one-way ANOVA followed by Tukey's post hoc testing. Comparisons between two groups were done by Student's t test with Welch's correction for unequal variances where appropriate.
The authors thank Monica Castanedes-Casey, Virginia Phillips, and Linda Rousseau for assistance with histopathology. Supported by the National Institutes of Health/National Institute on Aging grant (P01AG25531 to BAO, TEG).
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