Methylene blue modulates β-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice

Abstract

Amyloid precursor protein (APP) proteolysis is required for production of amyloid-β (Aβ) peptides that comprise β-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular β-amyloid deposits as well as levels of various Aβ species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, β-carboxyl-terminal APP fragment and β-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aβ production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.

Keywords: Alzheimer Disease; Amyloid; Amyloid Precursor Protein (APP); Animal Model; Anti-amyloidogenic; Methylene Blue; Secretase; Transgenic Mouse; β-Secretase.

FIGURE 1.

Oral methylene blue treatment completely reverses behavioral impairment in PSAPP mice. Data were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with MB (PSAPP-MB, n = 12) and also wild-type mice treated with vehicle (WT-V, n = 12) or with MB (WT-MB, n = 12) for 3 months beginning at 15 months of age and subjected to behavioral testing at 18 months of age. A, locomotion (upper panel) and rearing (lower panel) scores obtained from open field activity testing are shown. B, recognition index (%) in the object recognition test is shown from training (upper panel) and retention test phases (lower panel). C, Y-maze test data are represented as total arm entries (upper panel) and spontaneous alternation (lower panel). D, 2-day radial arm water maze test data are shown with five blocks per day for errors (upper panel) and escape latency (lower panel). All statistical comparisons were performed among PSAPP-V, PSAPP-MB, WT-V, and WT-MB mice (*, p < 0.05; **, p < 0.01 for PSAPP-V versus PSAPP-MB, WT-V, and WT-MB mice).

FIGURE 2.

Amelioration of cerebral parenchymal β-amyloid deposits in PSAPP mice treated with methylene blue. Representative photomicrographs were obtained from PSAPP mice treated with vehicle (PSAPP-V) or with MB (PSAPP-MB) for 3 months starting at 15 months of age (mouse age at sacrifice = 18 months) as well as 15-month-old PSAPP mice (PSAPP-15M). Immunohistochemistry using an anti-Aβ_17–24 monoclonal antibody (4G8) is depicted, demonstrating cerebral β-amyloid deposits in PSAPP-V, PSAPP-MB, and PSAPP-15M mice. Brain regions shown include the following: RSC (top), H (middle), and EC (bottom). In images from PSAPP-V and PSAPP-MB mice as well as PSAPP-15M mice, each right-hand panel is a higher magnification image from left panel insets. Scale bars, 200 μm (gray) and 100 μm (black).

FIGURE 3.

Cerebral parenchymal and vascular β-amyloid deposits and Aβ levels are reduced in methylene blue-treated PSAPP mice. Data were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with MB (PSAPP-MB, n = 12) for 3 months commencing at 15 months of age (mouse age at sacrifice = 18 months) for A–D as well as 15-month-old PSAPP mice (PSAPP-15M), n = 8 for A. A, quantitative image analysis for Aβ burden using an anti-Aβ_17–24 monoclonal antibody (4G8) is shown, and each brain region is indicated on the x axis (RSC, H, and EC). B, morphometric analysis of cerebral parenchymal β-amyloid deposits is shown in PSAPP-V and PSAPP-MB mice. Coronal brain sections were stained with 4G8 antibody, and deposits were blindly counted based on maximum diameter and assigned to one of three mutually exclusive categories as follows: small (<25 μm; top), medium (between 25 and 50 μm; middle), or large (>50 μm; bottom). Mean plaque subset number per mouse is shown on the y axis, and each brain region is represented on the x axis. C, severity of cerebral amyloid angiopathy (mean CAA deposit number per mouse) is shown on the y axis with brain region indicated on the x axis. D, TBS-soluble, 2% SDS-soluble, and 5 m guanidine HCl-extractable fractions from three-step extracted brain homogenates were separately measured by sandwich ELISA for human Aβ_1–40 and Aβ_1–42. Statistical comparisons for A–D are within each brain region and/or Aβ species, and between PSAPP-V and PSAPP-MB mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 4.

Methylene blue modulates amyloidogenic amyloid precursor protein processing via inhibition of β-site APP-cleaving enzyme 1 in PSAPP mice. A, Western blots are shown using an amino-terminal APP polyclonal antibody (pAb N-APP; holo-APP is shown) or a carboxyl-terminal BACE1 polyclonal antibody (pAb BACE1). An amino-terminal amyloid-β_1–17 (Aβ) monoclonal antibody (mAb 82E1), which detects various amyloidogenic APP cleavage fragments, including Aβ monomer and oligomers as well as phospho-C99 (P-C99) and nonphospho-C99 (C99), is also shown. Actin is included as a loading control for each blot, and densitometry data are represented as ratios of actin below each lane. B, densitometry analyses are shown for ratios of C-99 or P-C99 to actin. C, in the 2% SDS-soluble brain homogenate fraction, Aβ oligomers were measured by sandwich ELISA. D, densitometry analysis is shown for the ratio of BACE1 to actin. Representative Western blots for A were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 3) or with MB (PSAPP-MB, n = 3). Data for B–D were obtained from PSAPP mice treated with vehicle (PSAPP-V, n = 12) or with MB (PSAPP-MB, n = 12) for 3 months beginning at 15 months of age. All statistical comparisons are between PSAPP-V and PSAPP-MB mice (*, p < 0.05).

FIGURE 5.

Inhibition of amyloidogenic precursor protein metabolism in CHO/APP_WT cells by methylene blue. A, Aβ_1–40 and Aβ_1–42 species were separately measured in cell supernatants from CHO/APP_WT cells by sandwich ELISAs. B, inhibition of amyloidogenic APP processing in CHO/APP_WT cells treated with various doses of MB. Western blots using an amino-terminal Aβ_1–17 monoclonal antibody (mAb 6E10) or a carboxyl-terminal BACE monoclonal antibody (mAb BACE) show holo-APP, carboxyl-terminal fragment generated by amyloidogenic APP cleavage (C99, β-CTF), and BACE, respectively. β-actin is included as an internal reference control, and densitometry data are shown as ratios of β-actin below each lane. C, densitometry results are shown as ratios of C99 to β-actin at various MB doses. All statistical comparisons are between 0 μm and various doses of MB (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and similar results were observed in 3–4 independent experiments.

FIGURE 6.

Methylene blue modulates BACE1 expression and activity in CHO/APP_WT cells. A, modulation of BACE1 expression in CHO/APP_WT cells treated with various doses of MB. Western blots are shown using a carboxyl-terminal BACE monoclonal antibody (mAb BACE), a nicastrin polyclonal antibody (pAb nicastrin), a carboxyl-terminal PS1 monoclonal antibody (pAb PS1), and an amino-terminal presenilin enhancer 2 (PEN-2) polyclonal antibody (pAb PEN-2). β-actin is included as an internal reference control, and densitometry data are shown as ratios of β-actin below each lane. B, densitometry results are shown as ratios of BACE to β-actin at various MB doses. C, cell-free BACE1 activity assay results are displayed, and % of BACE1 activity is shown on the y axis. All statistical comparisons are between 0 μm and various doses of MB (*, p < 0.05; **, p < 0.01; ***, p < 0.001), and similar results were observed in 3–4 independent experiments.