Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

Abstract

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

Figure 1. MB treatment reduces tau forms in organotypic slice cultures. Slice cultures from JNPL3 mice (n = 6 mice per condition, three mice shown) were treated with either DMSO vehicle (C, control) or 0.02 μM MB (T, treated). (A) shows the results of immunoblotting with an antibody recognizing human tau (CP27) on the total and sarkosyl insoluble, aggregated tau fractions. The total protein fraction was also assayed for levels of tau phosphorylated at epitopes ser199, ser262, ser422, and ser396/404. Images were quantified and the chemiluminescence signal from MB treated hemibrain slices was expressed as percentage of signal from control treated hemibrain from the same animal (signal ± SE) (B). Total tau levels were unchanged with MB treatment. MB significantly reduced (p < 0.05) the level of sarkosyl insoluble, aggregated tau. Significant reduction was seen in tau hyperphosphorylated at ser199, ser262, ser422 and ser396/404 (phospho-tau epitope normalized to total tau). *p < 0.05.

Figure 2. MB treatment modulates autophagy markers in organotypic slice cultures. Samples utilized in Figure 1 were assayed for levels of autophagy markers. (A) shows the results of immunoblotting with antibodies against p62, cathepsin D (catD), BECN1 and LC3. Tubulin (tub) is shown as loading control. (B) shows the results quantified. 0.02 μM MB produced a significant decrease in p62 levels and an increase in catD, BECN1 and LC3-II signal relative to control treated slices **p < 0.01.

Figure 3. MB promotes AV formation and maturation. Primary neuronal cultures from GFP-LC3 mice were exposed to DMSO vehicle (A) or 0.02 μM MB (B) for 6 h. (C) shows quantification of the results. MB treated cells (gray bar) had significantly more puncta compared with vehicle-treated cells (black bar). In addition, the number of puncta per cell was greater following MB treatment (D). An additional group of primary neurons were incubated with 0.02 μM MB or DMSO control for 6 h. Following treatment, media was exchanged and cells were maintained in culture for a further 4 h to allow the MB to wash out. No significant difference was observed in the number of puncta in vehicle treated (E) vs. MB treated (F) cells. CHO cells transfected with a GFP-RFP-LC3 construct were treated with vehicle (G), 1 μM bafilomycin (H) or 0.01 μM MB (I). GFP and RFP images were collected and the percentage of GFP+/RFP+ positive AVs (yellow bars) and RFP+/GFP- positive autophagolysosomes (red bars) per cell were determined (J). MB treated cells had a significantly higher percentage of RFP+/GFP- puncta relative to control. Bafilomycin A₁ treated cells in contrast had a significantly lower percentage of RFP only puncta *p < 0.05, **p < 0.01.

Figure 4. Tau colocalizes with AVs. Primary neuronal cultures from GFP-LC3 mice were incubated with aggregated human tau for 18 h prior to fixation and labeling with fluorescently tagged antibody CP27 (human tau). (A) shows a confocal slice at 0.3 μm planar resolution indicating the green immunofluorescence signal from GFP-LC3; (B) indicates the red immunofluorescence signal from human tau aggregates. (C) shows the merged images (yellow) indicating that tau can colocalize with autophagic vacuoles, illustrated in the enlarged inset shown in (D). Human tau in AVs is indicated by the arrow.

Figure 5. BECN1 knockdown eliminates the effects of MB on tau. CHO cells expressing the BECN1 shRNA lentivirus (BECN1 LV), or control lentivirus (control LV) were treated with either vehicle (control) or MB for 6 h. BECN1 shRNA-expressing cells showed a significant decrease in BECN1 levels, and LC3-II (A) compared with cells expressing the control lentivirus. In cells expressing control virus, MB treatment resulted in a significant reduction in the level of tau (p < 0.001) (B). In contrast, cells in which BECN1 had been attenuated did not show reduced tau with MB treatment (C).

Figure 6. MB induces autophagy in CHO cells. Tau-transfected CHO cells were incubated for 6 h with vehicle or 0.01 μM MB (n = 6 per treatment). (A) shows immunoblots probed with antibodies against total tau, p62, BECN1 and LC3. (B) shows quantification of the immunoblots. Cells treated with MB (gray bars) had significantly reduced tau levels compared with vehicle-treated (black bars) cells, while the levels of BECN1 and LC3-II were increased. No effect on p62 levels was seen at this time point. All bars represent average ± SE, *p < 0.05.

Figure 7.

Kinase phosphorylation is altered with MB treatment. CHO cells treated with MB or rapamycin, a known autophagy inducer, were immunoblotted for kinase phosphorylation to assess activation status. To test whether MB acted in a similar way to rapamycin, cells were incubated with vehicle, rapamycin or MB for 6 h. (A) shows immunoblots for antibodies listed. (B) shows the quantification. The ratio between phosphorylated kinase at a relevant epitope and total kinase was determined in control (black bars), rapamycin (gray bars) and MB (hashed bars). MB significantly reduced levels of phospho-mTOR ser2448. Rapamycin also reduced levels of phospho-mTOR ser2448. Both rapamycin and MB reduced levels of phospho-p70 thr389 and phospho-IRS ser302. Rapamycin and MB also produced a significant increase in phospho-Akt ser473 and phospho-GSK-3β ser9. All bars represent average values and error bars SEM, *p < 0.05.

Figure 8. MB concentrates in the brain and reduces tau levels in treated mice. Cerebella were collected from MB and vehicle-treated animals and were subjected to LC–MS analysis (A). Animals given 0.02 mg/kg MB per day had an average 0.7 ± 0.1 μg MB per 100 mg of tissue. Mice in the 2 and 20 mg/kg per day groups had an average brain concentration of 1.73 ± 0.3 and 7.99 ± 0.78, respectively. All bars represent average values and error bars SEM. Male JNPL3 mice (n = 10 per group) were given 0.02mg/kg/day MB or water vehicle by daily oral gavage for two weeks. Brains were collected and fractionated to produce total, and sarkosyl insoluble (aggregated) tau. (B) shows immunoblots from these fractions probed for total tau and the following phosphorylated tau epitopes—ser199, ser202, thr231/ser235, ser396/404 and ser422. (C) shows the chemiluminescence signal (CU) quantified and expressed as the average per group ± SE. MB (gray bar) treatment resulted in lower total tau compare with control (black bar) (**p < 0.01). No significant change was observed in the levels of sarkosyl insoluble tau (data not shown). MB treatment led to a significant decrease in the levels of tau phosphorylated at ser199, ser202, thr231/ser235, and ser422 (*p < 0.05, **p < 0.01). Levels were normalized to total tau and expressed as an average ± SE.

Figure 9.

MB alters kinase activity and biomarkers of autophagy and lysosomes. Similar to the cell study shown in Figure 7, levels of total and phosphorylated epitopes of mTOR, p70S6K, Akt and GSK-3β were determined by immunoblotting (A). The ratio between the corresponding phosphorylated epitope and total kinase was determined, for vehicle (black bars) and MB treated (gray bars) animals (B). MB treatment results in a significant reduction of the phospho-mTOR ser2448 to mTOR ratio. The ratio of phospho-mTOR ser2481 and phospho-p70 thr389 (to total protein) unchanged. The ratio of phospho-Akt ser473 and phospho-GSK-3β ser9 (to total protein) was significantly increased with MB treatment. Levels of p55 thr199 and p85 thr458, regulatory subunits of PI3 kinase C1 were also significantly increased relative to control levels. (C) shows immunoblots prepared from JNPL3 mice probed for autophagy markers p62, cathepsin D, BECN1 and LC3. (D) shows quantification of the immunoblots. Treatment with 0.02 mg/kg MB (gray bars) led to a significant change in several markers of autophagy relative to vehicle-treated animals (black bars) including p62, BECN1 and cathepsin D. LC3-II levels were significantly increased in MB treated animals relative to vehicle. For all, control and MB treated samples were run on the same blot. LC3-II samples were run on smaller gels but loading order is the same. Significance levels indicated by *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 10. Proposed mechanism by which MB could stimulate autophagy through mTOR and Akt. (1) Inhibition of mTOR induces autophagy directly but also reduces phosphorylation of IRS1. (2) Dephosphorylation of IRS-1 results in decreased degradation and increased binding to the IGF receptor, increasing the receptor’s basal activity level. (3) Higher basal IGF receptor activity leads to increased phosphorylation of the PIK3R3/p55 and PIK3R1/p85 regulatory subunits of PtdIns 3-kinase (CI), thus increasing its activity. (4) PI3 kinase (CI) phosphorylates Akt. This in turn phosphorylates GSK-3 at the inhibitory site, lowering its activity. (5) In addition, reduced GSK-3 activity can lead to increased Bif-1 interaction with BECN1. (6) The SH3GLB1-BECN1 complex activates PtdIns 3-kinase (CIII) through UVRAG, stimulating autophagy.