Methylene blue reduces aβ levels and rescues early cognitive deficit by increasing proteasome activity

Abstract

Promising results have emerged from a phase II clinical trial testing methylene blue (MB) as a potential therapeutic for Alzheimer disease (AD), where improvements in cognitive functions of AD patients after 6 months of MB administration have been reported. Despite these reports, no preclinical testing of MB in mammals has been published, and thus its mechanism of action in relation to AD pathology remains unknown. In order to elucidate the effects of MB on AD pathology and to determine its mechanism of action, we used a mouse model (3xTg-AD) that develops age-dependent accumulation of Aβ and tau and cognitive decline. Here, we report that chronic dietary MB treatment reduces Aβ levels and improves learning and memory deficits in the 3xTg-AD mice. The mechanisms underlying the effects of MB on Aβ pathology appears to be mediated by an increase in Aβ clearance as we show that MB increases the chymotrypsin- and trypsin-like activities of the proteasome in the brain. To our knowledge, this is the first report showing that MB increases proteasome function and ameliorates AD-like pathology in vivo. Overall, the data presented here support the use of MB for the treatment of AD and offer a possible mechanism of action.

Figure 1

Methylene blue (MB) rescues early learning and memory deficits in 3xTg‐AD mice. (A) Chemical structure of Methylene Blue. (B) Schematic of the treatment paradigm. Six‐month‐old 3xTg‐AD (n = 17) and NonTg (n = 13) mice were treated with MB (0.025% w/w) supplemented diet provided ad libitum for 16 weeks. Additionally, age‐ and sex‐matched 3xTg‐AD (n = 14) and NonTg (n = 10) mice were given the control diet. Mice were evaluated in the spatial reference version of the Morris water maze during weeks 8 and 16. (C) All groups improved over 5 days of training. However, escape latency was significantly reduced in the MB‐treated 3xTg‐AD mice compared with the 3xTg‐AD mice on the control diet (P < 0.05). (D–F) Reference memory, measured 24 h after the last training trial, was significantly improved in the MB‐treated 3xTg‐AD mice compared with 3xTg‐AD mice on the control diet. Notably, MB‐treated 3xTg‐AD mice performed as well as both NonTg groups. Data are presented as means ± standard error of the mean and were analyzed using one‐way analysis of variance following by post hoc Bonferroni test to determine individual differences in groups. *indicates P < 0.05.

Figure 2

Methylene blue (MB) does not significantly increase mitochondrial function. (A) Representative Western blots of protein obtained from a mitochondrial enriched fraction from brains of MB‐treated (n = 6) and control 3xTg‐AD (n = 5) mice and probed with COX‐IV and VDAC antibodies as a loading control. (B) Densitometric analysis of the blots revealed no significant change in the COX‐IV levels. (C) No changes in cytochrome c oxidase activity were observed in MB‐treated 3xTg‐AD mice (n = 17) compared with 3xTg‐AD mice (n = 12) on the control diet. (D) ATP levels were measured in the cytosolic fraction and no significant changes were observed in MB‐treated 3xTg‐AD mice (n = 17) compared with 3xTg‐AD mice on the control diet (n = 13). (E) Kinetic readings were taken to measure ATP production in the mitochondrial enriched fraction energized with glutamate and malate by luciferase assay. ATP production rates were not significantly changed in MB‐treated 3xTg‐AD mice (n = 15) compared with 3xTg‐AD mice on the control diet (n = 11). Data were analyzed using t‐test analysis.

Figure 3

Methylene blue (MB) reduces Aβ levels in 3xTg‐AD mice. (A–B) MB significantly decreased soluble Aβ₄₂ and Aβ₄₀ levels as measured by enzyme‐linked immunosorbent assay. Data obtained from 12 3xTg‐AD on MB and 7 3xTg‐AD on the control diet. (C–D) Representative microphotographs depicting CA1 pyramidal neurons of MB‐treated and untreated 3xTg‐AD stained with 6E10 antibody. Notably, these results were also confirmed with Aβ42 specific antibodies. Sections from five different 3xTg‐AD mice on MB were compared with sections from five different 3xTg‐AD on the control diet. Inserts represent higher magnification views of panels C and D. Data are presented as ± standard error of the mean and analyzed by t‐test analysis. * indicates P < 0.05.

Figure 4

Methylene blue (MB) does not affect tau pathology in 3xTg‐AD. (A) Representative Western blots of proteins extracted from brains of 3xTg‐AD mice and probed with human tau specific antibody HT7, phospho‐specific antibody AT270 (which recognize tau phosphorylated at Thr181) with β‐actin as a loading control. (B) Densitometric analysis of blots (normalized to β‐actin) indicates that MB has no effect on tau accumulation or phosphorylation. Data obtained from five treated and five untreated 3xTg‐AD mice. (C–D) Representative microphotographs of CA1 pyramidal neurons stained with anti‐tau antibody HT7 indicate that MB does not alter tau mislocalization. Five mice/treatment were analyzed. Data are presented as ± standard error of the mean and analyzed by t‐test analysis.

Figure 5

Methylene blue (MB) increases proteasome activity. (A) Representative Western blots of proteins extracted from brains of MB‐treated and control 3xTg‐AD mice probed with antibodies 6E10, CT20 and β‐actin as a loading control. (B) Densitometric analysis of blots (normalized to β‐actin) indicates that the steady‐state levels of APP and the two major c‐terminal fragments (C99 and C83) were not affected by MB treatment. Data obtained from five treated and five untreated 3xTg‐AD mice. (C) Representative Western blots of proteins extracted from brains of MB treated and control 3xTg‐AD mice probed with different autophagy markers. (D) Densitometric analysis indicates that MB does not significantly affect the steady‐state levels of Beclin‐1 (Bec‐1), ATG‐7 or LC3B. Data obtained from five treated and five untreated 3xTg‐AD mice. (E–G) Brain homogenates from MB‐treated (n = 12) and control (n = 6) 3xTg‐AD mice were analyzed for proteasome activity. The data show that MB increases the chymotrypsin‐ and trypsin‐like activity, although it has no effect on the peptidylglutamyl‐peptide hydrolyzing (PDPH) activity. Data are presented as means ± standard error of the mean and analyzed by t‐test analysis. *indicates P < 0.05.