Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

https://doi.org/10.1016/j.bmc.2018.05.005Get rights and content

Abstract

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Introduction

Mitochondrial dysfunction and oxidative damage are hallmarks of numerous neurodegenerative diseases such as Friedreich’s ataxia (FRDA), as well as Alzheimer’s and Parkinson’s diseases.1, 2, 3, 4, 5 FRDA is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of a GAA trinucleotide repeat in the first intron of the FXN gene, impairing expression levels of fully functional frataxin protein by formation of sticky DNA and triple helices.6, 7, 8, 9, 10 FXN protein levels in individuals with FRDA are inversely correlated with the GAA repeat-burden in the FXN gene.7, 9 A large number of GAA repeats is associated with lower FXN protein levels, an earlier age-of-onset of FRDA and a faster rate of disease progression.9 Frataxin is encoded in the nucleus, expressed as a precursor polypeptide in the cytoplasm and imported into mitochondria with an important role in respiratory function and iron homeostasis.11, 12, 13, 14, 15, 16 FRDA is strongly associated with reduced synthesis of this mitochondrial iron chaperone, leading to mitochondrial iron accumulation, dysfunction of mitochondrial Fe-S containing enzymes (in particular mitochondrial respiratory complexes I, II and III as well as mitochondrial and cytosolic aconitases17) and increased Fenton-mediated free radical production.1, 4

Methylene blue (MB) is a redox active agent with a long history as a therapeutic agent. Clinical applications have included treatment of malaria, cyanide poisoning, methemoglobinemia and ifosfamide-induced encephalopathy.18, 19, 20, 21 MB is widely recognized as being neuroprotective in many neurodegenerative diseases such as Alzheimer’s disease.22 As a redox active agent, MB is able to cycle readily between its oxidized and reduced forms, enabling redirection of electrons to the mitochondrial electron transport chain, thereby enhancing adenosine triphosphate (ATP) production.23, 24, 25, 26 It has been shown that MB can act as an alternative electron carrier rerouting electrons from NADH to cytochrome c.23, 24, 25, 26 This process bypasses complex I/III blockage under pathological conditions and should theoretically ameliorate ROS production from the mitochondrial electron transport chain. Even during complex I inhibition by rotenone, MB can bypass electron transport chain blockage at complexes I and III, promoting respiration.23, 24, 25, 26 Methylene blue has been shown to rescue heart defects in a frataxin depleted drosophila model of FRDA.27 Further analysis of MB derivatives in this model indicated that only compounds with electron carrier properties (redox active), were able to prevent heart dysfunction. Therefore, the ability of MB analogues to employ alternative mitochondrial electron transfer mechanisms may constitute a better therapeutic strategy for the treatment of FRDA.

In earlier studies involving methylene violet and other heterocyclic antioxidants as potential mitochondrial therapeutic agents, we found that the presence of a lipophilic substituent was essential to activity, presumably because interaction with mitochondrial respiratory complexes embedded in the inner mitochondrial membrane represent the locus of action.26, 28 Presently we describe seven lipophilic MB analogues, some of which are capable of increasing frataxin levels and mitochondrial biogenesis, in addition to improving mitochondrial function and supporting ATP synthesis in FRDA lymphocytes.

Section snippets

Synthesis of the methylene blue analogues

In continuation of our previous research work on modifying parent compound MB to enhance biological activity, we have synthesized several new lipophilic MB derivatives (Fig. 1). The syntheses of MB analogues 17, having long alkyl substituents on the phenothiazine redox core (Fig. 1), were carried out using a Wittig reaction as the key step. The synthesis of the lipophilic MB analogues began with the protection of the N atom at position 10 of commercially available 2-cyanophenothiazine (Scheme 1

Conclusions

In conclusion, seven lipophilic MB analogues were synthesized and evaluated for their biological activities in vitro. Compound 5, lacking any cytotoxicity, was more effective and exhibited better antioxidant activity than MB, as well as superior activity in most of the other bioassays. The mechanism of the upregulation of FXN expression by MB and MB analogues requires further study. However, improvement in the quantity of mitochondria in FRDA patient-derived cells by treatment with MB and the

General experimental procedures

Human mitochondrial disease cell lines, Friedreich’s ataxia lymphocytes (GM16216 and GM15850) and clinically unaffected B-lymphocytes cell lines (GM16213 and GM15851) were obtained from Coriell Cell Repositories (Camden, NJ). Lymphocytes were routinely cultured in RPMI-1640 medium (Gibco, Life Technologies, Grand Island, NY) with added 15% fetal calf serum (FBS) and 1% penicillin–streptomycin antibiotic supplement (Cellgro, Manassas, VA). Cells were passaged every other day to maintain them in

Acknowledgment

This work was supported by a research grant from the Friedreich’s Ataxia Research Alliance.

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