N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease

Highlights

• NAC and vitamin E rescue short lifespan in gas-1(fc21) complex I mutant C. elegans.

• Several antioxidants partially rescue lifespan but improve healthspan in gas-1(fc21) worms.

• Vitamin E, and partially NAC, prevents brain death in complex I inhibited D. rerio.

• NAC improves cell viability in fibroblasts from complex I ND5 disease human subject.

• Cellular OXIDATIVE stress is a highly relevant treatment target in mitochondrial disease.

Abstract

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the “cellular antioxidant pathway”, they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease.

Introduction

Identification of effective therapies for primary (genetic-based) mitochondrial respiratory chain (RC) disease has been limited by an incomplete understanding of the leading pathogenic factors that underlie individual disorders [1]. Primary mitochondrial dysfunction causes a wide spectrum of clinical diseases, with features variably involving neurodevelopmental, myopathic, cardiac, renal, hepatic, gastrointestinal, endocrine, hearing and vision problems, as well as broader metabolic dysfunction [2]. Although the molecular mechanisms responsible for the pathogenesis of the diverse array of mitochondrial RC dysfunction-mediated diseases vary, oxidative stress is recognized to be a common element in their pathophysiology [3,4]. Oxidative stress can be induced by a range of mechanisms, including the generation of reactive oxygen species (ROS) that damage DNA, proteins, and lipids, as well as the depletion of endogenous antioxidant systems [5]. Multiple antioxidant therapies have long been used in the clinical care of mitochondrial disease patients as an empiric means to reduce oxidative stress [6], with additional strategies under development to enhance antioxidant delivery specifically within the mitochondrial compartment [7,8]. However, it has been unclear whether antioxidants that target oxidative stress throughout the entire cell or primarily within mitochondria are necessary or sufficient to restore cellular and organism health [9]. We postulate that more globally directed antioxidant therapies may be required in primary mitochondrial RC disease to effectively boost endogenous defenses and reduce the secondary oxidative stress that impairs many aspects of cellular function [10].

It is unknown whether antioxidants with different mechanisms of action have potential therapeutic advantanges at the level of cellular, organ, and overall individual health in primary mitochondrial RC disease [[11], [12], [13]]. A range of antioxidants variably used to empirically treat RC disease patients include coenzyme Q10 (CoQ10), vitamin E, vitamin C, and lipoic acid [11,14], with several other agents proposed largely in the basic research setting such as N-acetylcysteine (NAC), orotic acid, and mitochondrial-targeted coenzyme Q10 (MitoQ) [[15], [16], [17], [18], [19]]. However, there exists no widely accepted means to clinically monitor antioxidant treatment efficacy either on oxidative stress burden in different tissues or at the more integrated outcomes level of overall patient health. Furthermore, it is not known whether there are optimal doses, or possible toxicities, that may result from antioxidant therapy use in mitochondrial disease patients, either from off-target effects or from their potentially negative impact on physiologic oxidant levels that are essential for intracellular signaling [20]. While clinical trials are the gold-standard means to test a purported therapy's impact in human disease at the level of survival, feeling, and function, comparative analysis of all possible antioxidant therapies, without some pre-clinical means to prioritize lead therapeutic candidates, is neither a practical nor efficient approach in a rare disease cohort such as primary RC disease.

Pre-clinical modeling to prioritize therapeutic leads can enable deeper insights into these key questions [21], where antioxidants of predicted benefit in human RC disease can be objectively evaluated by mechanistic studies in simple animal and cellular models of primary RC dysfunction [10,22,23]. Here, C. elegans was used as the primary model animal system in which to systematically investigate the preventative (treatment from early development) and therapeutic (treatment beginning on first day of adulthood) effects of 7 antioxidant treatments that have been empirically used, or rationally proposed, for the clinical treatment of human RC disease (Fig. 1). The primary model studied was the well-established C. elegans gas-1(fc21) model of RC complex I disease, which results from a homozygous p.R290K missense mutation in the nuclear-encoded complex I NDUFS2 subunit ortholog [24]. These mutant worms have been shown to have 70% decreased complex I-dependent respiratory capacity [25], significantly shortened lifespan at 20 °C [25], multiple secondary alterations in intermediary metabolic pathways identifiable by genome-wide expression analysis, free amino acid profiling [26] and stable isotope based flux analysis [27], and increased mitochondrial oxidant burden [28]. Here, we systematically quantified the physiologic effects and mechanisms of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MitoQ, or MS010), lipoic acid (lipoate), and orotic acid (orotate) on gas-1(fc21) RC mutant animals' lifespan as the primary outcome. Secondary mechanistic analyses of treatment effects were performed on their integrated metabolism at multiple levels to assess for potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome, and intermediary metabolic flux. Antioxidant therapies that showed the greatest benefit in C. elegans mitochondrial complex I disease mutant worms were subsequently validated in a zebrafish RC complex I disease brain death model, and in human fibroblast cells from mitochondrial complex I disease patients.