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Keywords:

  • γ-secretase modulator;
  • Aβ;
  • Alzheimer's disease;
  • amyloid plaque;
  • APP transgenic mouse;
  • cognition

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Given that amyloid-β 42 (Aβ42) is believed to be a culprit in Alzheimer's disease (AD), reducing Aβ42 production should be a potential therapeutic approach. γ-Secretase modulators (GSMs) cause selective reduction of Aβ42 or both reduction of Aβ42 and Aβ40 without affecting total Aβ through shifting the γ-cleavage position in amyloid precursor protein. We recently reported on GSM-2, one of the second-generation GSMs, that selectively reduced brain Aβ42 level and significantly ameliorated cognitive deficits in plaque-free 5.5-month-old Tg2576 AD model mice. Here, we investigated the effects of GSM-2 on 10-, 14-, and 18-month-old mice which had age-dependent increase in amyloid plaques. Eight-day treatment with GSM-2 significantly ameliorated cognitive deficits measured by Y-maze task in the mice of any age. However, GSM-2 reduced brain soluble Aβ42 only in 10-month-old mice. In contrast, GSM-2 markedly reduced newly synthesized soluble Aβ42 in both 10- and 18-month-old mice with similar efficacy when measured using the stable isotope-labeling technique, suggesting that nascent Aβ42 plays a more significant role than plaque-associated soluble Aβ42 in the cognitive deterioration of Tg2576 mice. These findings further indicate the potential utility of approach to reducing Aβ42 synthesis in AD therapeutic regimens.

Abbreviations used
AD

Alzheimer's disease

APP

amyloid precursor protein

amyloid-β peptide

GSI

γ-secretase inhibitor

GSM

γ-secretase modulator

LC-MS/MS

liquid chromatography-tandem mass spectrometry

TBS

tris-buffered saline

According to the amyloid hypothesis, amyloid-β peptide of 42 amino acids (Aβ42) should be a culprit in Alzheimer's disease (AD) (Hardy and Selkoe 2002). γ-Secretase is the enzyme responsible for the final step of Aβ production from amyloid precursor protein (APP) (De Strooper et al. 1998; Wolfe et al. 1999). Although γ-secretase inhibitors (GSIs) reduce total Aβ production, they cause accumulation of APP-β-carboxyl-terminal fragment (β-CTF or C99) and inhibition of other substrate processing such as Notch. These characteristics may be associated with the failure of phase III trials with semagacestat (Siemers et al. 2011). Recently, clinical development of another GSI, avagacestat, has also been discontinued in the middle of phase II stage (http://www.bms.com/news/features/2012/Pages/AvagacestatDevelopmentStatus.aspx).

γ-Secretase modulators (GSMs) reduce the production of only Aβ42 or both Aβ42 and Aβ40 without affecting total Aβ by shifting the γ-cleavage position in APP (Weggen et al. 2001, 2003; Hall et al. 2010; Kounnas et al. 2010). GSMs are expected to overcome the defects of GSIs, and the concept has been currently under evaluations in humans. We recently reported that GSM-2, one of the second-generation GSMs, selectively reduced Aβ42 production and ameliorated cognitive deficits in plaque-free 5.5-month-old APP transgenic Tg2576 mice where semagacestat and avagacestat failed to reverse cognitive deficits (Mitani et al. 2012). However, the effects of GSM-2 on plaque-rich aged APP mice, that are more similar to a clinical setting, remain to be studied.

Although the brain amyloid plaque composed of insoluble Aβ fibrils is a pathological hallmark of AD, soluble Aβ levels correlate more with disease severity or synaptic destruction in AD (Lue et al. 1999; McLean et al. 1999). A recent report suggested that amyloid plaques could be another source of soluble Aβ, particularly Aβ42, in APP transgenic mice (Hong et al. 2011). If in fact plaque-derived soluble Aβ42 plays a significant role in memory deficit, then GSMs should be ineffective at ameliorating AD deficits, as they can reduce only nascent Aβ.

To address this issue, we determined the effects of GSM-2 on the cognitive deficits in Tg2576 mice of ages bearing amyloid plaques. A previous report showed that brain amyloid plaques in Tg2576 mice first appear at 8 months and expand at between 15 and 23 months to degrees similar to those found in AD patients (Kawarabayashi et al. 2001). Here, we subjected Tg2576 mice aged 10, 14, and 18 months to the Y-maze task for evaluating spatial working memory. We also investigated the effects of GSM-2 on the brain Aβ42 production rate using the stable isotope-labeling technique (Bateman et al. 2006; Castellano et al. 2011) to determine the role of newly synthesized Aβ42 in cognitive deterioration.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Drugs

GSM-2 ({(2S,4R)-1-[(4R)-1,1,1-trifluoro-7-methyloctan-4-yl]-2-[4-(trifluoromethyl)phenyl]piperidin-4-yl}acetic acid) and its enantiomer were synthesized in accordance with the patent application (Garcia et al. 2007). The chemical structures of these drugs are shown in Fig. 1. The drugs were dissolved in dimethylsulfoxide for in vitro studies and suspended in 0.5% methylcellulose for in vivo studies.

image

Figure 1. Chemical structures of γ-secretase modulator (GSM)-2 and its enantiomer.

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Cellular Aβ assay

SK-N-BE(2) human neuroblastoma cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were cultured in 96-well plates overnight and then treated with each drug at various concentrations in 0.5% dimethylsulfoxide for 6 h. Aβ1-42 levels in the media were measured using the 384-well plate-based sandwich-ELISA system constructed with 44A3 anti-Aβ42 monoclonal antibody (IBL, Gunma, Japan), biotin-labeled 82E1 anti-Aβ N-terminal monoclonal antibody (IBL), streptavidin-horseradish peroxidase conjugate (Invitrogen, Carlsbad, CA, USA), and TMB as the chromogen. Aβ1-40 levels in the media were measured using an ELISA kit (IBL). Cell viability was evaluated using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Fitchburg, WI, USA).

Animals

Tg2576 mice expressing human APP695 with the Swedish mutation (K670N/M671L) (Hsiao et al. 1996) were used as previously described (Mitani et al. 2012). We used female mice in this study, because male mice tended to fight and injure each other, which may potentially affect behavioral studies. Animals were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-approved facility in a temperature- and humidity-controlled colony room (maintained at 23 ± 3°C and 55 ± 15%, respectively) under a 12-h light/dark cycle with water and laboratory chow supplied ad libitum. All in vivo experimental procedures were performed during the light cycle. Procedures involving animals and their care were conducted in accordance with AAALAC guidelines and Astellas Pharma Inc. guidelines for the care and use of animals under approved protocols from the Institutional Animal Care and Use Committee of Astellas Pharma Inc.

Immunohistochemistry

Tg2576 mouse brains at age 6, 10, 14, and 18 months were immersion-fixed in 4% paraformaldehyde/phosphate buffer for 2 days at 4°C, then placed in 16% sucrose/phosphate buffer for 2 days at 4°C. The brains were frozen and cut into 20-μm-thick coronal sections with a cryostat and then incubated with a mixture of anti-Aβ42 and anti-Aβ40 polyclonal antibodies (diluted 1 : 1000; IBL) on free-floating overnight at room temperature. Aβ immunoreactivity was visualized using the Vector Elite horseradish-peroxidase ABC kit (Vector Laboratories, Burlingame, CA, USA) with 3,3′-diaminobenzidine as the chromogen.

Y-maze test

Spatial working memory in mice was evaluated by recording spontaneous alternation behavior in the Y-maze task as previously described (Mitani et al. 2012). Briefly, each drug was orally administered once daily for 8 days. Three hours after final administration, each animal was placed at the end of one arm of the apparatus and allowed to freely explore for 8 min. The alternation rate was defined as entries into all three arms on consecutive occasions using the following formula:

Alternation rate (%) = Number of alternations/(Number of total arm entries−2) × 100

Data were eliminated in cases where the number of total arm entries was less than 10.

Quantitation of hippocampal Aβ

Tris-buffered saline (TBS)-soluble Aβ in the hippocampus was quantified via ELISA as previously described (Mitani et al. 2012). Briefly, the hippocampus was isolated immediately after the Y-maze test followed by storage at −80°C. Frozen samples were homogenized in an ultrasonic homogenizer with 10-fold volume of TBS (Tris 25 mM, NaCl 137 mM, KCl 2.68 mM; pH 7.4) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) followed by ultracentrifugation at 100 000 g and 4°C for 1 h. Levels of Aβ1-42 and Aβ1-40 in the supernatant were measured using ELISA kits (IBL). To quantify insoluble Aβ, aliquots of the homogenate were solubilized with final 5 M guanidine hydrochloride for 1 h at 23 ± 3°C and applied to ELISA.

Stable isotope-labeling study

Newly synthesized Aβ in the Tg2576 mouse brain was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described (Bateman et al. 2006; Castellano et al. 2011) with some modifications. Mice, aged 10 and 18 months, were injected intraperitoneally with 300 mg/kg 13C6-labeled leucine (Cambridge Isotope Laboratories, Andover, MA, USA) dissolved in saline at 20 mg/mL followed by the oral administration of 10 mg/kg GSM-2 or vehicle. Brains were harvested 2 h after injection and hemisected, including removal of the olfactory bulb and cerebellum, followed by snap freezing with liquid nitrogen and storage at −80°C. Frozen hemispheres were weighed and homogenized in a teflon homogenizer with 1 mL TBS containing 1% Triton X-100 and Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) on ice followed by ultracentrifugation at 100 000 g and 4°C for 1 h. Small aliquots of supernatant were subjected to Aβ x-42 ELISA (Wako, Osaka, Japan).

The remaining supernatant was incubated with 5 μg of 44A3 anti-Aβ42 monoclonal antibody and Protein G-Sepharose 4 fast flow beads (GE Healthcare, Uppsala, Sweden) for 16 h at 4°C, then separated using spin columns. Small aliquots of the solution separated from the beads were subjected to Aβ x-40 ELISA (Wako), and the remaining solution was immunoprecipitated with 5 μg of anti-Aβ40 polyclonal antibody (IBL). All beads that trapped either Aβ42 or Aβ40 were washed with TBS and 25 mM ammonium bicarbonate. Aβ was eluted with 1,1,1,3,3,3-hexafluoroisopropanol, then dried and re-suspended with 25 μL of 20% acetonitrile in 25 mM ammonium bicarbonate followed by digestion with 1 μg Sequencing Grade Modified Trypsin (Promega) for 18 h at 37°C.

Samples were then subjected to an API 2000 electrospray ionization-tandem mass spectrometer (Applied Biosystems, Carlsbad, CA, USA) automatically tuned with a synthetic Aβ17-28 peptide, LVFFAEDVGSNK (Peptide Institute, Osaka, Japan), and coupled to an Agilent 1100 high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA). Samples were injected into (10–20 μL each) and separated using a CAPCELL PAK C18, 3 μm particle, 2.0 × 35 mm column (Shiseido, Tokyo, Japan) at a flow rate of 0.5 mL/min and with a gradient mixture of solvents A (5% acetonitrile and 0.1% formic acid in water) and B (5% water and 0.1% formic acid in acetonitrile), using the following program: 10% B for 1 min, 10% to 30% B in 0.1 min, 30% B for 2 min, 30 to 90% B in 0.1 min, and 90% B for 2.5 min.

Aβ17-28 tryptic peptide and its 13C6-leucine-labeled form were monitored with multiple reaction monitoring (MRM) of m/z ([M + 2H]2+) 663.5 [RIGHTWARDS ARROW] 213.3 and 666.5 [RIGHTWARDS ARROW] 219.4, respectively. The 13C6-labeling rate was calculated from the peak height of each peptide form, since the peak height of the synthetic peptide was proportional to its amount in the range 2–10 000 fmol (r2 = 0.999), which covered all sample measurements. Newly synthesized Aβ amount was calculated using the following formula:

Newly synthesized Aβ amount = ELISA measured Aβ amount × 13C6-labeling rate.

Statistical analysis

Data are presented as mean ± SEM or individual plots, and all statistical analyses were conducted using SAS software (SAS Institute, Cary, NC, USA). The Dunnett's multiple comparison test was used for the concurrent studies of GSM-2 and the enantiomer. The unpaired Student's t-test was used for the study of only GSM-2 or comparison between Tg2576 group and wild-type group. For all tests, a value of p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

GSM-2 inhibited Aβ42 production in human neuroblastoma cells

We evaluated the effects of GSM-2 and its enantiomer on cellular endogenous Aβ production using SK-N-BE(2) human neuroblastoma cells. GSM-2 potently inhibited cellular Aβ42 production with an IC50 value of 0.045 μM. The enantiomer also inhibited Aβ42 production, but at a potency that was 28-fold lower than that of GSM-2, indicating that the biological activity of GSM-2 was chirally selective. In contrast, Aβ40 production was only slightly inhibited by GSM-2 and unchanged by the enantiomer (Fig. 2 and Table 1). Neither compounds affected cell viability at any of the tested concentrations (data not shown).

Table 1. Drug effects on Aβ production in human neuroblastoma cells
 IC50 (μM)
Aβ1-42Aβ1-40
  1. Values are shown as mean ± SEM for three independent experiments.

GSM-20.045 ± 0.005> 10
Enantiomer1.24 ± 0.17> 10
image

Figure 2. Effects of γ-secretase modulator (GSM)-2 and its enantiomer (Enan) on Aβ production in human neuroblastoma cells. SK-N-BE(2) cells were exposed to various concentrations of each drug for 6 h in 96-well plates. Aβ1-42 (left graph) and Aβ1-40 (right graph) secreted in the culture medium were measured via ELISA. Data are presented as mean ± SEM of three independent experiments. IC50 values are presented in Table 1.

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GSM-2 ameliorated cognitive deficits in Tg2576 mice at all ages tested

To determine whether plaque formation with aging affects GSM-2 efficacy, we examined Tg2576 mice aged 10, 14, or 18 months with different degrees of plaque presence in the brain. Although no brain amyloid plaques were observed in 6-month-old mice after staining with a mixture of anti-Aβ42 and anti-Aβ40 antibodies, plaques did begin to form by 10 months and expanded in degree in 14- and 18-month-old mice age-dependently (Fig. 3), findings consistent with a previous report (Kawarabayashi et al. 2001). We chose 10 mg/kg as the drug test dose based on findings in our acute-dose pilot study, in which this dose of GSM-2 showed a significant reduction in brain soluble Aβ42 in 10-month-old Tg2576 mice. GSM-2 or its enantiomer was orally administered for 8 days, and Y-maze tests were conducted 3 h after the final administration. Vehicle-treated Tg2576 mice at all ages tested showed significantly lower spontaneous alternation rates than their wild-type littermates, suggesting deficits in spatial working memory. These memory deficits were significantly ameliorated by treatment with GSM-2. In other words, plaque formation with aging did not affect GSM-2 efficacy. In contrast, the enantiomer failed to restore deficits in 10- and 14-month-old mice (18-month-old mice were not tested) (Fig. 4a), indicating that the cognition-improving effects of GSM-2 are because of its inhibitory activity on Aβ42 production. No significant changes in the number of total arm entries were found with drug treatment at any age (data not shown).

image

Figure 3. Age-related amyloid pathology in Tg2576 mice. Brain slices of Tg2576 mice aged 6, 10, 14, and 18 months were immunostained with a mixture of anti-Aβ42 and anti-Aβ40 antibodies. Amyloid plaques emerged from age 10 months and dramatically increased with age.

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image

Figure 4. Effects of γ-secretase modulator (GSM)-2 and its enantiomer (Enan) on cognitive deficits in Tg2576 mice. (a) Each drug was orally administered to Tg2576 mice aged 10, 14, and 18 months at 10 mg/kg for 8 days, after which animals were subjected to Y-maze tests. GSM-2 significantly ameliorated cognitive deficits at all ages tested, whereas the enantiomer had no effect (effects of the enantiomer in 18-month-old mice were not assessed). Dotted lines, chance level of alternation behavior. #p < 0.05; ##p < 0.01 by Student's t-test. **p < 0.01 compared with vehicle group by Dunnett's test. All data are presented as mean ± SEM (n = 6 or 7). (b) Correlations between alternation rate and brain soluble Aβ42 level were depicted as scatter plots. The hippocampus was harvested immediately after Y-maze tests and extracted with tris-buffered saline (TBS), and then subjected to ELISA. A statistically significant correlation was seen in 10-month-old mice but not in 14- and 18-month-old mice.

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GSM-2 reduced brain soluble Aβ42 levels in only 10-month-old Tg2576 mice

To corroborate the link between cognitive improvement and brain Aβ42 reduction, mice were killed immediately after the Y-maze tests, and TBS-soluble hippocampal Aβ levels were quantified using an ELISA. When the alternation rate was plotted against soluble Aβ42 level in each mouse, a statistically significant correlation was seen in 10-month-old mice (p = 0.007) but not in 14- and 18-month-old mice (Fig. 4b). Intergroup analysis revealed that GSM-2 significantly reduced soluble Aβ42 level by 34% compared to the vehicle group in 10-month-old mice, while the enantiomer did not. However, GSM-2 did not reduce soluble Aβ42 level in 14- and 18-month-old mice despite cognitive improvements. Basal levels of soluble Aβ42 were 5- and 12-fold higher in 14- and 18-month-old mice, respectively, than in 10-month-old mice, suggesting that short GSM-2 treatment does not reduce the increased Aβ42 levels that occur with aging (Fig. 5a). Further, neither drug reduced soluble Aβ40 levels at any age, consistent with our in vitro observations (Fig. 5b).

image

Figure 5. Effects of γ-secretase modulator (GSM)-2 and its enantiomer (Enan) on brain soluble Aβ levels in Tg2576 mice. The hippocampus was harvested immediately after Y-maze tests and extracted with tris-buffered saline (TBS), and then subjected to ELISA. (a) GSM-2 significantly reduced levels of soluble Aβ1-42 in 10- but not in 14- and 18-month-old mice, whereas the enantiomer had no effect. (b) Soluble Aβ1-40 levels were not reduced by either drug in animals of any age. **p < 0.01 compared with vehicle group by Dunnett's test.

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To rule out the possibility that changes in TBS-insoluble Aβ levels contributed to cognitive improvement, TBS-homogenate was solubilized with 5M guanidine and subjected to ELISA. Neither drug reduced guanidine-solubilized Aβ42 and Aβ40 levels at any age (Fig. 6). The basal level of guanidine-solubilized Aβ42 in 10-month-old mice was 62-fold higher than that of TBS-soluble Aβ42 and robustly increased with age (by 20- and 53-fold in 14- and 18-month-old mice, respectively).

image

Figure 6. Effects of γ-secretase modulator (GSM)-2 and its enantiomer (Enan) on brain insoluble Aβ levels in Tg2576 mice. Aliquots of hippocampal tris-buffered saline (TBS) homogenate were treated with guanidine hydrochloride and subjected to ELISA. Both insoluble Aβ1-42 (a) and Aβ1-40 (b) levels were unchanged by either drug in animals of any age.

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GSM-2 reduced levels of newly synthesized brain Aβ42 in both 10- and 18-month-old Tg2576 mice

To determine the effects of GSM-2 on brain Aβ42 production in Tg2576 mice, we applied the stable isotope-labeling technique which can discriminate newly synthesized and pre-existing Aβ. Tg2576 mice aged 10 or 18 months were intraperitoneally administered 300 mg/kg 13C6-leucine followed by oral administration of 10 mg/kg GSM-2 or vehicle. After 2 h, whole brains were collected, and brain soluble Aβ was extracted with TBS containing 1% Triton X-100 and quantified via ELISA (= total soluble Aβ42 or Aβ40). The extracts were then immunoprecipitated with anti-Aβ42 or anti-Aβ40 antibody and trypsinized. 13C6-labeled tryptic peptides of Aβ42 and Aβ40 were quantified via LC-MS/MS (= de novo Aβ42 or Aβ40).

Respective labeled/unlabeled ratios of Aβ42 and Aβ40 in vehicle-treated 10-month-old mice were 6.7 ± 0.6% and 6.9 ± 1.0% (mean ± SEM; n = 6), both of which decreased in 18-month-old mice (to 0.82 ± 0.13% and 2.1 ± 0.3%, respectively) because of an increase in levels of pre-existing unlabeled Aβ. Although GSM-2 significantly reduced levels of total soluble Aβ42 by 42% in 10- but not 18-month-old mice, the compound significantly reduced de novo levels of Aβ42 in both 10- and 18-month-old mice by 58% and 53%, respectively (Fig. 7). GSM-2 reduced neither total nor de novo Aβ40 at both ages. Given these findings, we concluded that brain Aβ42 production in 18-month-old Tg2576 mice was inhibited by GSM-2, an effect that may be associated with cognitive improvement.

image

Figure 7. Effects of γ-secretase modulator (GSM)-2 on newly synthesized brain Aβ levels in Tg2576 mice. 13C6-leucine and GSM-2 were administered to Tg2576 mice aged 10 and 18 months. (a) Brains were extracted with tris-buffered saline (TBS) containing 1% Triton X-100 and then subjected to ELISA (= total soluble Aβ). GSM-2 significantly reduced soluble Aβ x-42 in 10- but not in 18-month-old mice, whereas Aβ x-40 was unchanged in any age group. (b) Extracted brain samples were imunoprecipitated and trypsinized, and then subjected to LC-MS/MS analysis to quantify 13C6-leucine-labeled Aβ (= de novo soluble Aβ). GSM-2 significantly reduced de novo Aβ42 but not de novo Aβ40 in both age groups examined. **p < 0.01 compared with vehicle group by Student's t-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reducing Aβ levels using GSI, GSM, and inhibitors of β-site APP-cleaving enzyme 1 (BACE1) is believed to be a potential therapeutic regimen for AD, with several related drugs currently undergoing clinical trial. However, the Aβ-lowering effects of these compounds as well as their behavioral effects have thus far largely been demonstrated pre-clinically in plaque-free young APP transgenic mice, although prophylactic chronic treatment in these mice did show a positive effect on plaque prevention (Comery et al. 2005; Martone et al. 2009; Fukumoto et al. 2010; Kounnas et al. 2010; Chang et al. 2011; Van Broeck et al. 2011). In this study, we demonstrated that reducing Aβ42 production by GSM-2 improved cognitive deficits in Tg2576 mice even at ages when Aβ plaque burden was significant.

Given that cellular studies revealed that the biological activity of GSM-2 is chirally selective, as GSM-2 inhibited cellular Aβ42 production 28-fold more potently than did its enantiomer (Fig. 2), we decided to use the enantiomer as a negative control in subsequent behavioral studies. Findings in these behavioral studies showed that GSM-2 significantly ameliorated cognitive deficits in Tg2576 mice aged 10, 14, and 18 months, regardless of plaque levels, whereas the enantiomer showed no effect, suggesting that the observed behavioral benefits are because of reduction of Aβ42 synthesis (Fig. 4). In fact, while the enantiomer did not affect any Aβ levels, GSM-2 significantly reduced brain soluble Aβ42 by 34% in 10-month-old mice (Fig. 5a), an effect that was in agreement with in vitro potency and plasma ‘free’ drug level (0.028 μM), although brain ‘total’ drug level was much higher (2.3 μM). However, the ability of GSM-2 to reduce Aβ levels was not detected in 14- or 18-month-old mice (Fig. 5), an observation likely attributable to the robust increase in the basal level of soluble Aβ42 which occurs along with plaque development. In other words, any reduction of Aβ42 synthesis by GSM-2 is masked by age-related increases in levels of pre-existing Aβ42.

Therefore, to determine the actual reduction of Aβ42 synthesis achieved by GSM-2, we used the stable isotope-labeling technique, which allowed for quantification of newly synthesized Aβ independent of pre-existing Aβ (Bateman et al. 2006; Castellano et al. 2011). We demonstrated that GSM-2 significantly reduced levels of newly synthesized brain Aβ42 in both 10- and 18-month-old mice with similar efficacy (Fig. 7), suggesting that nascent Aβ42 may play a more significant role in cognitive impairment than pre-existing Aβ42, as GSM-2 ameliorated the impairment without reducing total soluble Aβ42 in 18-month-old mice, in which Aβ42 was mostly pre-existing Aβ42.

The reason for the distinct contributions of nascent and pre-existing Aβ42 on cognitive deterioration remains to be addressed. We speculate that while most or part of nascent Aβ42 is secreted into the synaptic cleft and affects synaptic function, most of the pre-existing soluble Aβ42 is non-synaptic. Although previous reports using Aβ microdialysis have suggested that soluble Aβ42 in plaque-rich brains could be dynamically equilibrated with insoluble Aβ pools in the form of plaques (Cirrito et al. 2003; Hong et al. 2011), the physiological impact of the plaque-associated soluble Aβ42 remains unknown. Our results provide some insights on the matter, although further studies are needed.

In conclusion, GSM-2 significantly ameliorated cognitive deficits in AD model mice even when treatment began after plaque development, indicating the potential utility of GSMs for AD therapeutic regimens albeit need for validation using other GSMs. Furthermore, the stable isotope-labeling technique enabled determination of the Aβ42-lowering effects of GSM-2 in plaque-rich brains, suggesting the usefulness of this technique for target-engagement in AD patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Dr. Shingo Yamasaki for his kind assistance throughout this study and his valuable input in the preparation of the manuscript. We declare no conflicts of interest in connection with this manuscript. All authors were employees of Astellas Pharma Inc. at the time of this study.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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