A Food and Drug Administration-approved asthma therapeutic agent impacts amyloid β in the brain in a transgenic model of Alzheimer disease

Abstract

Interfering with the assembly of Amyloid β (Aβ) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aβ-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aβ monomers into higher-order oligomers and fibrils in vitro without affecting Aβ production. In vivo, the levels of soluble Aβ are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aβ in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aβ economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.

Keywords: Aggregation; Alzheimer Disease; Amyloid β (AB); Cromolyn Sodium; asthma; in vivo microdialysis; mouse.

FIGURE 1.

Cromolyn sodium inhibits Aβ polymerization in vitro but does not impact pre-existing oligomers. A, structures of cromolyn Sodium and fisetin. B, left panel, representative curves of Thioflavin-T fluorescence increase upon Aβ fibrillization after addition of dimethyl sulfoxide (top panel) or cromolyn sodium (5, 50, and 500 nm, bottom panels). Fibrillization of synthetic Aβ₄₀ (left column) or Aβ₄₂ (right column) peptides was followed over 1 h. The corresponding V_max index (milliunits/minute) is indicated on each graph. Right panel, bar graphs summarizing Aβ₄₀ (top graph) and Aβ₄₂ (bottom graph) fibrillization, which is significantly decreased in presence of cromolyn sodium even though smaller effects were observed on the Aβ₄₂ fibrillization process. C, representative TEM pictures of Aβ₄₂ fibrils formed after 48-h incubation of synthetic Aβ₄₂ peptides (0.2 mg/ml) in the presence of 50 or 500 nm of cromolyn sodium. Scale bar = 100 nm. D, Aβ oligomerization assay using the split-luciferase complementation assay. HEK293 cells, which were engineered to stably express luci-Aβ₄₂ and ferase-Aβ₄₂, were incubated with 0, 10 nm, 10 μm, and 1 mm of cromolyn sodium for 12 h. The amounts of oligomers were measured using luciferase activity. E, oligomer dissociation assay using the split-luciferase complementation assay. Pre-existing oligomers composed of split-luciferase-conjugated Aβ were incubated with 0, 10 nm, 10 μm, and 1 mm of cromolyn sodium for 12 h. The amounts of oligomers were measured using luciferase activity. The ratio of luminescence after incubation with cromolyn sodium compared with PBS was calculated (n = 3 experiments). *, p < 0.05, **, p < 0.01; n.s., not significant.

FIGURE 2.

Cromolyn sodium reduces the concentration of soluble monomeric Aβ, not oligomeric Aβ, in the brain of APP/PS1 mice. A, one week after daily intraperitoneal injection with cromolyn sodium or PBS, TBS-soluble Aβ_x-40 (left panel) and Aβ_x-42 (right panel) were measured by ELISA with (black column) or without (white column) preincubation with Gdn-HCl. B, oligomeric Aβ content in TBS-soluble brain extracts was measured using 82E1/82E1 Aβ oligomer-specific ELISA. C, representative immunoblotting of TBS-soluble brain extracts with anti-Aβ antibody (6E10 and 82E1, left panel). The densitometry of Aβ monomers was quantified (right panel) (n = 3–5 mice/group). *, p < 0.05; **, p < 0.01; n.s., not significant.

FIGURE 3.

Cromolyn sodium lowers the levels of Aβ_x-40 and Aβ_x-42 in both Triton-X and SDS fractions of APP/PS1 brain extracts. As mentioned previously, AD transgenic mice were treated with PBS or escalating concentrations of cromolyn sodium (1.05, 2.1, and 3.15 mg/kg) for 1 week before euthanasia. A, bar graph representing the concentrations of Aβ_x-40 (left panel) and Aβ_x-42 (right panel) measured by ELISA in the Triton X extracts. B, bar graph representing the concentrations of Aβ_x-40 (left panel) and Aβ_x-42 (right panel) measured by ELISA in the SDS extracts (n = 3–5 mice/group). *, p < 0.05.

FIGURE 4.

Cromolyn sodium does not alter the levels of insoluble Aβ peptides and amyloid deposition in APP/PS1 mice after a week of treatment. A, cromolyn sodium was intraperitoneal injected into 7.5-month-old APP/PS1 mice daily for 1 week with three different doses (1.05, 2.1, and 3.15 mg/kg). One week after daily injection, insoluble Aβ_x-40 (white column) and Aβ_x-42 (black column) present in the formic acid fraction of brain extracts were measured using Aβ ELISA. n.s., not significant. B, percentages of Aβ_x-40 and Aβ_x-42 in each biochemical fraction (TBS, Triton, SDS, and formic acid (FA)) according to the total level of Aβ. Distributions were compared between control transgenic mice and animals treated with 1.05, 2.1, and 3.15 mg/kg of cromolyn sodium for a week. No significant changes could be detected. C, representative pictures of the amount of amyloid deposits in mice treated with PBS or cromolyn sodium (3.15 mg/kg) for 1 week (using the rabbit anti-human amyloid β (N) antibody from Immuno-Biological Laboratories). Scale bars = 2 mm (top panels) and 200 μm (bottom panels). D, bar graphs summarizing the amyloid load (top panel) and plaque density (bottom graph) in both group (n = 3–5 mice/group).

FIGURE 5.

Acute treatment with cromolyn sodium decreases the levels of monomeric Aβ_x-40, not oligomeric Aβ, in the ISF of APP/PS1 mice. A, cromolyn sodium was injected intraperitoneally into 9-month-old APP/PS1 mice daily for 1 week at the 3.15 mg/kg dose. One week after daily injection, ISF sample were collected by in vivo microdialysis. The concentrations of Aβ_x-40 (left panel) and Aβ_x-42 (right panel) in ISF were measured by ELISA. B, oligomeric Aβ in ISF was quantified using the 82E1/82E1 Aβ oligomer-specific ELISA assay (n = 5 mice/group). *, p < 0.05; n.s., not significant.

FIGURE 6.

Cromolyn sodium decreases the half-life of Aβ in ISF. A, representative decay Aβ_x-42 curves in mice treated with PBS and cromolyn sodium (3.15 mg/kg) after infusion of 100 nm of γ-secretase inhibitor (compound E) by reverse microdialysis. ISF samples were collected every hour before and after γ-secretase inhibitor perfusion. The concentration of ISF Aβ_x-42 was measured by ELISA after preincubation with Gdn-HCl. B, bar graph summarizing the extrapolated Aβ_x-42 half-life 1 week after intraperitoneal injection with cromolyn sodium or PBS (n = 5 mice/group). *, p < 0.05.

FIGURE 7.

Cromolyn sodium does not affect the levels of Aβ in the plasma but promotes microglial Aβ clearance. A, quantification of the plasmatic levels of Aβ_x-40 and Aβ_x-42 1 week after treatment with PBS or escalating doses of cromolyn sodium (n = 3–5 mice/group). B, representative images of localization of amyloid deposits (6E10, green) and microglia (Iba1, red) in mice treated with cromolyn sodium (3.15 mg/kg) or PBS daily for 7 days. The percentage of amyloid occupied by Iba1-positive processes was calculated for each deposit and showed an increased overlap between Aβ and Iba1 after treatment with cromolyn sodium (n = 3 mice for PBS and n = 5 mice for cromolyn sodium; approximately 20 plaques were evaluated for each animal). Scale bar = 10 μm. C, effect of cromolyn sodium on microglial Aβ uptake in vitro. Microglial cells were cultured and incubated with 50 nm of synthetic Aβ₄₀ or Aβ₄₂ and 0, 10 nm, 10 μm, or 1 mm of cromolyn sodium for 16 h. After the incubation, the concentrations of Aβ_x-40 (left panel) and Aβ_x-42 (right panel) in media were measured using Aβ ELISA and normalized with microglia cell number and according to the PBS control condition (n = 3 experiments). *, p < 0.05, **, p < 0.01; n.s., not significant.