We carried out animal studies using the well-characterized APPSwedish-expressing Tg2576 mice. 3-month old Tg2576 mice were acclimatized for 2 months and then randomly assigned to a total of 7 treatment groups, including 6 drug-treatment groups and one vehicle-treatment group (Table 1). Each group received one dose of cromolyn (1.0 or 3.15 mg/kg), ibuprofen (0.5 or 2.0 mg/kg), cromolyn plus ibuprofen (n = 8–10/group), or PBS (vehicle; n = 10). The drugs were prepared in sterile PBS and administered by IP, based on 0.1 mL/30 g body weight. The treatments were performed 3 times per week for 12 weeks in total. The body weight was measured 2 weeks after the initial treatment and once per week afterwards. We found that all treatments were well tolerated, and there were no significant differences in body weight among the groups throughout the treatment period (Supplementary Figure 1). All treated mice were sacrificed at 8-month old and tissues were harvested and processed for post-mortem analysis.

Table 1 Experimental groups and treatments. Full size table

Effects of Cromolyn and Ibuprofen on Aβ levels in Tg2576 Mice

We first investigated whether cromolyn, ibuprofen, or cromolyn plus ibuprofen influenced brain Aβ levels in the TBS-insoluble (formic acid extracted) protein fraction. In the TBS-insoluble fraction, both low and high-dose cromolyn almost completely abolished both Aβ40 and Aβ42 species (p < 0.001; vs. vehicle; Fig. 1A; Supplementary Figure 2A), while robustly increasing Aβ38 levels. Specifically, low-dose cromolyn decreased Aβ40 and Aβ42 levels by 92.4% (p < 0.001) and 94.8% (p < 0.001), respectively, and increased Aβ38 levels by 402.0% (p < 0.05) compared to vehicle. High-dose cromolyn decreased Aβ40 and Aβ42 levels by 98.7% (p < 0.001) and 99.6% (p < 0.001), respectively, and increased Aβ38 levels by 191.6% as compared to vehicle. We also found that low and high-dose ibuprofen alone decreased both Aβ40 and Aβ42 levels (p < 0.001 and p < 0.05, respectively; Fig. 1A; Supplementary Figure 2A), while robustly increasing Aβ38 levels (p < 0.001 for the high-dose). Combined low-dose cromolyn and ibuprofen also led to a significant decrease in Aβ40 and Aβ42 levels (p < 0.001 and p < 0.05, respectively; vs. vehicle; Fig. 1A; Supplementary Figure 2A) and a marked increase in Aβ38 levels (p < 0.05; vs. vehicle; Fig. 1A; Supplementary Figure 2A). Finally, combined high-dose cromolyn and ibuprofen displayed a significant decrease in both Aβ40 and Aβ42 levels (p < 0.05 and p < 0.001, respectively; vs. vehicle; Fig. 1A), but with no significant change in Aβ38 levels as compared to vehicle (Fig. 1A; Supplementary Figure 2A).

Figure 1 Cromolyn and/or ibuprofen robustly affected Aβ levels and Aβ42:Aβ40 ratios in brain TBS-insoluble samples. (A,B) MesoScale Aβ-triplex analyses were applied to brain TBS-insoluble samples. Differences in Aβ levels (A) and Aβ(42:40) ratios (B) were assessed comparing various treatment groups to vehicle. Mean ± SEM; n ≥ 8 (Table 1); *p < 0.05; ***p< 0.001 (vs. vehicle); one-way ANOVA with Dunnett’s multiple comparison test. Abbreviations: Aβ(42:40): Aβ42: Aβ40. Full size image

We next analyzed the effects of cromolyn and ibuprofen, alone, or in combination, on the Aβ42:Aβ40 ratio in brain TBS-insoluble fractions as compared to vehicle. Neither cromolyn nor ibuprofen, alone, affected Aβ42:Aβ40 ratios vs. vehicle (Fig. 1B; Supplementary Figure 2B). Interestingly, combined low-dose cromolyn with ibuprofen led to an increased Aβ42:Aβ40 ratio (p < 0.05; vs. vehicle; Fig. 1B; Supplementary Figure 2B). However, this did not appear to be dose-dependent since combined high-dose cromolyn with ibuprofen did not significantly affect the Aβ42:Aβ40 ratio vs. vehicle (Fig. 1B; Supplementary Figure 2B).

We next investigated the effects of cromolyn and ibuprofen, alone, or in combination on brain TBS-soluble protein fractions. Neither cromolyn, nor ibuprofen alone, signicantly affected Aβ levels, although high-dose cromolyn increased levels of soluble Aβ42 (by 126.0%; p < 0.05; Fig. 2A; Supplemental Figure 2C), as well as the soluble Aβ42:Aβ40 ratios (Fig. 2B; Supplemental Figure 2D). In addition, combined high-dose cromolyn and ibuprofen significantly increased levels of soluble Aβ40 and Aβ38 levels (p < 0.05; Fig. 2A; Supplemental Figure 2C). With regard to effects of cromolyn and ibuprofen, alone, or in combination on Aβ42:Aβ40 ratios in TBS-soluble fractions, high-dose cromolyn, alone, significantly increased Aβ42:Aβ40 ratios as compared to vehicle (by 40.0%; p < 0.01; Fig. 2B; Supplemental Figure 2D).

Figure 2 High dose cromolyn and combined high dose cromolyn with ibuprofen upregulated Aβ levels in brain TBS-soluble samples; High dose cromolyn elevated Aβ42:Aβ40 ratios in brain TBS-soluble samples. (A,B) MesoScale Aβ-triplex analyses were applied to brain TBS-soluble samples. Differences in Aβ levels (A) and Aβ42:Aβ40 ratios (B) were analyzed comparing various treatment groups to vehicle. Mean ± SEM (normalized to vehicle); n ≥ 8 (Table 1); *p < 0.05; **p < 0.01 (vs. vehicle); one-way ANOVA with Dunnett’s multiple comparison test. Full size image

We next investigated whether cromolyn and ibuprofen, alone, or in combination, affected Aβ levels in plasma (Fig. 3A,B; Supplementary Figure 3A,B) and CSF (Fig. 4A,B; Supplementary Figure 3C,D), as compared to vehicle. Only low-dose cromolyn, alone, increased the plasma Aβ42:Aβ40 ratios (p < 0.05; Fig. 3B; Supplementary Figure 3B). However, this effect was not dose-dependent since high-dose cromolyn, alone, did not exhibit similar effects.

Figure 3 Low dose cromolyn increased plasma Aβ42:Aβ40 ratios. (A,B). MesoScale Aβ-triplex analyses were applied to plasma samples. Differences in Aβ levels (A) and Aβ42:Aβ40 ratios (B) in plasma were analyzed comparing various treatment groups to vehicle. Mean ± SEM; n ≥ 8 (Table 1); *p < 0.05 (vs. vehicle), one-way ANOVA with Dunnett’s multiple comparison test. Full size image

Figure 4 Cromolyn and/or ibuprofen did not significantly influence the levels of Aβ and Aβ42:Aβ40 ratios in mouse CSF samples. (A,B) MesoScale Aβ-triplex analyses were applied to CSF samples. Differences in Aβ levels (A) and Aβ42:Aβ40 ratios (B) were analyzed comparing various treatment groups to vehicle. Mean ± SEM; n ≥8 (Table 1); one-way ANOVA with Dunnett’s multiple comparison test. Full size image

Overall, we showed that cromolyn and ibuprofen, alone, and/or in combination with each other, decreased levels of cerebral TBS-insoluble Aβ40 and Aβ42, and increased soluble brain or plasma Aβ species and Aβ42:Aβ40 ratios (cromolyn, alone). In summary, these findings are suggestive of an anti-aggregation/pro-clearance mechanism of cromolyn and ibuprofen, alone, or in combination with each other on cerebral Aβ depositon.

Effects of Cromolyn and Ibuprofen on Microglia and Aβ

Acute treatment with cromolyn for one week was previously shown to lead to an increased number of microglia around β-amyloid plaques6. We therefore performed a stereological analysis to determine if similar effects could be observed after chronic exposure (12 weeks beginning at 5 months old) with cromolyn and ibuprofen, alone, or in combination with each other. Brains in the vehicle group displayed minimal β-amyloid burden, consistent with previous reports in the Tg2576 mouse model at this age, and no significant changes in the β-amyloid plaque density (Supplementary Figure 4A) or β-amyloid load (Supplementary Figure 4B) were observed across the treatment groups. These findings are likely explained by the fact that 8 month old Tg2576 mice exhibit minimal plaque pathology.

We next assessed the percentage of β-amyloid deposits that overlapped with microglial processes. As shown in the representive images of colocalization of β-amyloid deposits (detected by the Bam10 antibody, green) and microglia (detected by the Iba1-specific antibody, red) (Fig. 5A), the percentage of β-amyloid deposits occupied by Iba1-positive processes was calculated for each deposit in all animals. Low-dose cromolyn (p < 0.05; vs. vehicle; Fig. 5B) increased the overlap of microglial processes with β-amyloid deposits, while high dose cromolyn exhibited only a trend in this direction. No changes in overlap were observed after treatment with the low or high doses of ibuprofen (Fig. 5B). Combined low-dose and high-dose cromolyn with ibuprofen also significantly increased the overlap of β-amyloid deposits with microglial processes as compared to ibuprofen alone (p < 0.01; Fig. 5B). Overall, our results in the 12-week treatment period were consistent with the previous acute 7-day study suggesting that microglial recruitment and subsequent clearance of Aβ is increased by cromolyn. Treatment with ibuprofen, alone or in association with cromolyn, had no impact on this parameter.

Figure 5 Cromolyn, but not ibuprofen, increased microglial activity in AD mice. (A,B) Cromolyn increased microglial activity related to β-amyloid deposits in mice. Iba1-positive microglial processes (red) colocalizing with β-amyloid deposits (green) were showed (A) and quantified (B). We represented pictures of β-amyloid deposits and Iba-1 positive microglia for the highest concentration only from each drug treatment group. Mean ± SEM; *p < 0.05, **p < 0.01, one-way ANOVA with post-hoc Tukey’s multiple comparison test. (C) Cromolyn, but not ibuprofen, promoted Aβ42 uptake in BV2 microglial cell culture studies. BV2 microglial cell cultures were treated with cromolyn and/or ibuprofen (10 μM, 100 μM, 1 mM) for 16 hours. Afterwards, cells were incubated with soluble Aβ42 and the compounds for additional 3 hours. After incubation, cells were collected for Aβ ELISA analysis. BV2 microglial cells treated with cromolyn (100 μM, 1 mM), and with combined cromolyn and ibuprofen (100 μM, 1 mM for each compound) exhibited increased Aβ42 uptake levels relative to BV2 microglia treated with vehicle. Mean ± SEM; n = 3; **p < 0.01, ***p < 0.001, one-way ANOVA post-hoc Tukey’s multiple comparison test. Full size image

To further explore the effects of cromolyn and ibuprofen, alone, or in combination, we next employed a microglial cell-based assay to assess effects on Aβ42 uptake. Specifically, BV2 murine microglial cell cultures were treated with cromolyn and/or ibuprofen (10 μM, 100 μM and 1 mM, respectively) for 16 hours. Subsequently, cells were incubated with soluble Aβ42 and the compounds for 3 hours. After incubation, cells were collected for ELISA analysis to measure Aβ42 levels. BV2 microglial cells treated with cromolyn, alone (100 μM, 1 mM), and with cromolyn plus ibuprofen (100 μM, 1 mM for each compound), exhibited increased Aβ42 uptake relative to cells treated with vehicle (n = 3; p < 0.001 and p < 0.01, respectively, one-way ANOVA with Tukey’s test; Fig. 5C). In contrast, BV2 microglial cells treated with ibuprofen (10 μM, 100 μM, 1 mM), alone, displayed no change in Aβ42 uptake levels relative to cells treated with vehicle. Collectively, the microglial cell-based studies were consistent with the results of the Tg2576 mouse-based studies. Moreover, the Tg2576 mouse and cell-based studies support the notion that cromolyn decreased TBS-insoluble Aβ levels at least, partially, by promoting a microglial pro-phagocytic state that leads to enhanced clearance of Aβ.