Methylene Blue Modulates Huntingtin Aggregation Intermediates and Is Protective in Huntington's Disease Models

Department of Biological Chemistry, Psychiatry and Human Behavior, University of California, Irvine, California 92697, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 08/2012; 32(32):11109-19. DOI: 10.1523/JNEUROSCI.0895-12.2012
Source: PubMed

ABSTRACT Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

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Available from: Emily Mitchell Sontag, Nov 24, 2014
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    • "Methylene blue (MB) 1 has been used with multiple therapeutical purposes in the treatment of malaria [3] [30] [41], carbon monoxide or cyanide poisoning [23], methemoglobinemia [47], and neurodegenerative diseases [16] [21] [25] [66] [72] [73] [87] [96] and in photodynamic therapy [58]. MB is able to take electrons on its aromatic thiazine ring to be reduced to leukomethylene blue (MBH 2 ) and transfer electrons to other compounds depending on the redox states and the concentration of MB (see [65]). "
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    ABSTRACT: Summary The redox dye, methylene blue (MB) is proved to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100nM, 300nM and 1μM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea-pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by High Resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with either substrate (glutamate-malate, succinate or alpha-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm and increased the rate of Ca(2+)-uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry in all conditions; both in resting, ATP-synthesizing and respiration-impaired mitochondria with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2, without superoxide formation, observed in the presence of MB is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB.
    Free Radical Biology and Medicine 09/2014; 77. DOI:10.1016/j.freeradbiomed.2014.09.024 · 5.74 Impact Factor
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    • "The mechanism of action of this compound is unknown, but it has been speculated that it could act as antitau aggregation agent. Some studies theorize that the compound blocks aggregation of not only tau but also several other proteins such as α-synuclein, TDP-43, or even Aβ (Sontag et al., 2012), whereas other studies find the compound to be ineffective (Van Bebber et al., 2010). Other suggestions for methylene blue mode of action were autophagy stimulation (Congdon et al., 2012) or enhancement of proteasome activity (Medina et al., 2011). "
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    ABSTRACT: Peering into the field of Alzheimer's disease (AD), the outsider realizes that many of the therapeutic strategies tested (in animal models) have been successful. One also may notice that there is a deficit in translational research, i.e., to take a successful drug in mice and translate it to the patient. Efforts are still focused on novel projects to expand the therapeutic arsenal to "cure mice." Scientific reasons behind so many successful strategies are not obvious. This article aims to review the current approaches to combat AD and to open a debate on common mechanisms of cognitive enhancement and neuroprotection. In short, either the rodent models are not good and should be discontinued, or we should extract the most useful information from those models. An example of a question that may be debated for the advancement in AD therapy is: In addition to reducing amyloid and tau pathologies, would it be necessary to boost synaptic strength and cognition? The debate could provide clues to turn around the current negative output in generating effective drugs for patients. Furthermore, discovery of biomarkers in human body fluids, and a clear distinction between cognitive enhancers and disease modifying strategies, should be instrumental for advancing in anti-AD drug discovery.
    Frontiers in Pharmacology 06/2014; 5:146. DOI:10.3389/fphar.2014.00146 · 3.80 Impact Factor
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    • "The same homogenates used in the AGERA assays were also analyzed for SDS-insoluble mutant HTT by filter-retardation assay as previously described [62] [63] [64]. 50 ␮g of homogenate were diluted in 0.1% SDS and filtered through cellulose acetate membrane (Schleicher & Schuell, 0.2 ␮m pore size) using a dot blot filtration apparatus (Bio-Rad) and washed using 0.1% SDS. "
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    ABSTRACT: Background: Some promising treatments for Huntington's disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1–548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.
    Journal of Huntington's disease 01/2013; 2(1-1):47-68. DOI:10.3233/jhd-130001
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