Neurometabolic mechanisms for memory enhancement and neuroprotection of methylene blue

Abstract

This paper provides the first review of the memory-enhancing and neuroprotective metabolic mechanisms of action of methylene blue in vivo. These mechanisms have important implications as a new neurobiological approach to improve normal memory and to treat memory impairment and neurodegeneration associated with mitochondrial dysfunction. Methylene blue's action is unique because its neurobiological effects are not determined by regular drug-receptor interactions or drug-response paradigms. Methylene blue shows a hormetic dose-response, with opposite effects at low and high doses. At low doses, methylene blue is an electron cycler in the mitochondrial electron transport chain, with unparalleled antioxidant and cell respiration-enhancing properties that affect the function of the nervous system in a versatile manner. A major role of the respiratory enzyme cytochrome oxidase on the memory-enhancing effects of methylene blue is supported by available data. The memory-enhancing effects have been associated with improvement of memory consolidation in a network-specific and use-dependent fashion. In addition, low doses of methylene blue have also been used for neuroprotection against mitochondrial dysfunction in humans and experimental models of disease. The unique auto-oxidizing property of methylene blue and its pleiotropic effects on a number of tissue oxidases explain its potent neuroprotective effects at low doses. The evidence reviewed supports a mechanistic role of low-dose methylene blue as a promising and safe intervention for improving memory and for the treatment of acute and chronic conditions characterized by increased oxidative stress, neurodegeneration and memory impairment.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Chemical structure and redox balance of methylene blue

The tri-heterocyclic thiazide ring of MB allows the presence of a delocalized positive charge that confers a high reduction potential. At the same time, the presence of imine groups (C = N − R) confers high antioxidant activity to the MB molecule. In its oxidized form, methylene blue (MB) accepts electrons from an electron donor (XH2). In its reduced form, leucomethylene blue (MH2) is colorless, acts as an electron donor, and it can transfer electrons to oxygen to form water. In vivo and at low concentrations methylene blue and leucomethylene blue are at equilibrium, so that they form a reversible reduction-oxidation system. The auto-oxidizing capacity of MB provides a mechanism for electron transfer to oxygen, which accounts for its antioxidant and metabolic-enhancing properties, as well as its hormetic dose-response effects at the biochemical, physiological and behavioral levels.

Figure 2. Inverted U-shaped curve typical of hormesis

Increasing doses induce stimulatory or beneficial effects. Maximal stimulation is seen at intermediate doses and corresponds to 30-60% increases compared to control, as opposed to several fold-increases typical of linear-non-threshold dose-response curves. As the dose increases, the biological response becomes less stimulatory and can be no different than control. With even higher doses, inhibitory or toxic effects are observed. This hormetic dose-response is also called the β curve. Behavioral and neurochemical hormetic effects of methylene blue have been described in vivo (Bruchey and Gonzalez-Lima, 2008).

Figure 3. Mitochondrial mechanism of action of methylene blue in memory enhancement and neuroprotection

Normally, electron donors (NADH and FADH2) reduce mitochondrial complex I or II. Electrons (e−) are subsequently transferred to ubiquinone (CoQ), complex III, cytochrome c (Cyt c) and complex IV. As this electron transfer occurs in a tightly regulated fashion, the energy released from each redox reaction is used to pump protons (H+) into the intermembrane space to generate an electrochemical gradient that is used to activate the enzyme ATP synthase. Methylene blue (MB) is a synthetic chemical compound and does not occur endogenously. However, in optimal conditions MB can emulate the activity of endogenous electron carriers within the inner mitochondrial membrane. MB is an auto-oxidizable compound that becomes readily available to mitochondria, where it can be reduced to leucomethylene blue (MBH2). In physiological conditions characterized by high energy demands, MB enters a reversible redox cycle, increasing cytochrome oxidase (complex IV) activity, interacting with oxygen to form water and thus facilitating cell respiration. During the oxidizing conditions prevailing during excessive energy demands or mitochondrial failure, low concentrations of MB exert antioxidant and electron shuttling actions that support the respiratory chain function. At high concentrations, methylene blue can take electrons away from the electron transport chain complexes, thereby impairing their activity.

Figure 4. Memory enhancement with methylene blue

Methylene blue (MB) improved memory retention examined in a holeboard spatial memory task, in which rats used spatial cues to learn the location of sweetened cereal placed in different holes (Callaway et al., 2004). A) MB USP 1 mg/kg was given daily after learning sessions using a consistent baiting pattern (pattern 1). Solid circles represent baited holes. Memory retention was subsequently tested in an unbaited probe trial. After the unbaited trial, the baiting pattern was reversed (reversal), and the animals were tested again after re-learning the task. B) The graph shows mean (± standard error bar) of memory performance (% visits to correct holes) in groups of vehicle control and MB-treated subjects. The MB group had twice as many correct responses as the control group for the first baiting pattern (pattern 1), the second baiting pattern (Reversal), and the averaged total (Overall). *p = 0.037, **p = 0.000014, ***p = 0.00017

Figure 5. Activity-dependent effects of methylene blue

A) MB acts on pre-synaptic and postsynaptic mitochondria (Mit) and will preferentially accumulate in neurons within activated networks with high energy demands and active mitochondria. This generalized but at the same time activity-dependent enhancing effect is explained by the existence of elevated proton gradients in highly active mitochondria, which drive the location of MB within the cell. B) MB enhances the activity of the electron transport chain and modulates nitric oxide synthase (NOS). NOS is normally activated by glutamatergic receptors (AMPAR and NMDAR) to produce nitric oxide, which acts as a second messenger and, among other actions, regulates cytochrome oxidase activity, thus exerting a strong influence on respiration. Metabolically active mitochondria communicate with the nuclear and mitochondrial genome (red arrows) to up-regulate all cytochrome oxidase (COX) and some nitric oxide synthase (Nos1), NMDA receptor (Grin 1 and Grin 2b) and AMPA receptor (Gria2) subunit genes, all under the influence of the nuclear respiratory transcription factor NRF-1. C) Thus, gene expression orchestrated by NRF-1 allows for co-localization of proteins critical for energy metabolism and synaptic transmission that aid in synaptic strengthening and improved memory function.

Figure 6. The network effects of MB relevant to visuospatial memory

Patients with mild cognitive impairment show early impairments in visuospatial memory. A) The diagram shows key structures of the circuit of Papez, a neuroanatomical network involved in the processing of visuospatial memory. Increases in metabolic activity in both the thalamus and hippocampus induce increased metabolic activity in the posterior cingulate cortex (solid arrows). The posterior cingulate cortex is a region considered as a major “hub” in the neural network for visuospatial function, receiving and sending numerous projections. B) Hypometabolism in the posterior cingulate cortex (cross) induces a network disruption in which high metabolic activity in the thalamus and hippocampus induces a further decrease (dashed arrow) in activation of the posterior cingulate. This disruption is behaviorally evident as a subtle visuospatial memory retrieval deficit. C) Methylene blue concentrates in areas with high metabolic activity and facilitates energy metabolism. By doing this, MB facilitates the adaptive changes in connectivity within the thalamus and hippocampus, which are represented by the patchwork pattern shown in these regions. Such changes consist of tendencies of subregions to act as either functional clusters or independent functional nodes. This internal subregional reconfiguration allows for the recruitment of additional brain regions that can “make up” for the faulty connectivity and mediate an appropriate behavioral output. These network effects possibly occur in addition to the direct local neuroprotective effects exerted by MB in those regions originally affected by hypometabolism (Riha et al., 2011).

Figure 7. Neuroprotection with methylene blue

MB prevented the neurodegeneration induced by the neurotoxin rotenone in the brain (Rojas et al., 2009b). The figure is a Nissl-stained coronal section of the forebrain of a rat receiving bilateral intrastriatal infusions of rotenone alone (left hemisphere) and rotenone plus MB (right hemisphere). The rotenone-treated hemisphere showed a large area of neurodegeneration, characterized by a cavity of liquefactive necrosis surrounded by a rim of reactive gliosis (asterisk). This lesion was accompanied by an enlarged lateral ventricle (black arrow). In contrast, the MB co-treated hemisphere showed significantly less damage, with a comparatively smaller lesion limited to the corpus callosum (white arrow). Similar dramatic neuroprotective results were found in other experiments with rats and mice that used bilateral or unilateral infusions of rotenone as compared to rotenone plus MB in the eye (Zhang et al., 2006; Rojas et al., 2009a) or in the brain (Rojas et al., 2009b).