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

Keywords: Antioxidant; C. elegans; Fibroblasts, genetic disease; Mitochondria; Therapeutic modeling; Zebrafish.

Figure 1. Schematic experimental overview of antioxidant drug treatments in three RC complex I disease models

Effects of 7 antioxidant drugs having different sites and/or purported modes of action were systematically studied in the C. elegans complex I deficient gas-1(fc21) mutant worm at the level of animal lifespan and physiologic mechanisms. Leading treatments showing maximal lifespan benefit were subsequently validated by assessing their effects on survival and/or mitochondrial physiology in RC complex I disease human fibroblast cells and/or a rotenone model of complex I inhibition in zebrafish. NAC, N-Acetylcysteine. ROS, reactive oxygen species. MitoQ, mitochondrial-targeted CoQ10. ETC, electron transport chain. FACS, Fluorescence-activated cell sorting. HPLC, high performance liquid chromatography. GC/MS, gas chromatography/mass spectrometry. Vit, vitamin.

Figure 2. Antioxidant drugs variably rescue short lifespan of complex I mutant gas-1(fc21) C. elegans

All drug studies were performed at 20°C compared to buffer-only treated gas-1(fc21) and wild-type N2 Bristol worms, with effects of exposure tested from initiation in development at L1 stage (‘Dev’) or at initiation upon reaching adulthood (‘young adult, YA’). The complex I mutant strain, gas-1(fc21) has significantly shortened median and maximal lifespan compare to N2 (see panels A–H). (A) N-acetylcysteine [NAC, 2.5 mM] significantly rescued median lifespan (31% increase relative to untreated gas-1(fc21) worms) when treatment was started during early development, and 19% when started at the young adult stage (p<0.0001 and p<0.0001, respectively). (B) Vitamin E [250 μM] increased both maximal and median lifespan by up to 33% when started at the developmental stage and 23% when started at the young adult stage (p<0.0001 and p<0.0001, respectively). (C) CoQ10 [650 uM] also rescued both maximal and median lifespan (14%) in both developmental and young adult stage treatment (p<0.005 and p<0.03 respectively). (D) Lipoic acid [10 μM] significantly increased lifespan (p<0.0003) when it was administered during development, there was a milder effect when worms were treated in young adult hood (p<0.04). (E) Orotic acid increased both median and maximal lifespan administered in 50 μm concentration during development (p<0.02). (F) Vitamin C in 1mM concentration has a mild beneficial effect both in developmental and young adult treatment on maximal lifespan (p<0.04 and p<0.006 respectively). (G) MS010 [‘MitoQ’, 10 uM] failed to significantly increase the median lifespan in both developmental and young adult age (p>0.05). (H) dTTP [10 μM] was used as a negative control for MS010.

Figure 3. Antioxidant drugs variably rescue mitochondrial pathophysiology of complex I mutant gas-1(fc21) C. elegans

In vivo fluorescence analysis of relative mitochondrial oxidant burden, membrane potential, and mitochondrial content in gas-1(fc21) mutants treated with antioxidant drugs was performed relative to buffer-only treated gas-1(fc21) and wild-type worms. Specifically, mitochondrial content, mitochondrial oxidant burden, and mitochondrial membrane potential were microscopically assessed by in vivo terminal pharyngeal bulb (PB) relative fluorescence quantitation using MitoTracker Green FM (MTG), MitoSOX (SOX), or TMRE, respectively. Effects of each drug were compared to buffer control (i.e., gas-1(fc21) with either 0.1% ethanol for Vitamin E, or S. basal for other six water-soluble drugs), with control data pooled as effects of 0.1% ethanol were negligible compared to S. basal solvent. Significant differences in the mean fluorescence intensity between strains under different experimental conditions was assessed by mixed-effect ANOVA, which accounts for potential batch effect due to samples being experimentally prepared, processed, and analyzed on different days by including a batch random effect in the model. Statistical significance threshold was set at P < 0.05 and all statistical analyses were performed in SAS 9.3. P-value conveys the significance of the difference between untreated N2 and untreated gas-1(fc21) (strain effect) or the difference between gas-1(fc21) plus drug and untreated gas-1(fc21) (treatment effect). For each parameter, each drug treatment assay was repeated in 3 to 10 independent trials, with n=50 worms per trial. Bars and error bars convey mean +/− SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus concurrent gas-1(fc21) buffer control.

Figure 4. Antioxidant drug effects on upregulated antioxidant pathway in gas-1(fc21) adult worms

Transcriptome expression changes in the gene ontology (GO) defined antioxidant (GO:0016209)) pathway were evaluated in gas-1(fc21) mitochondrial complex I mutant nematodes treated for 24 hours during early adulthood with one of (A) six water-soluble drugs relative to S. basal control or (B) lipophilic vitamin E relative to ethanol control. Wild-type worms (N2 Bristol) were treated only with buffer control. Each bar indicates comparative pathway changes in second group listed as compared to first. Similar level reduction in the GO-defined defense pathway was seen in the young adult treated worms with each antioxidant therapy tested except CoQ10. Enrichment score conveys degree to which upregulated antioxidant pathway in gas-1(fc21) relative to N2 control were normalized by each treatment in gas(fc21) worms.

Figure 5. Antioxidant drug effects on upregulated KEGG pathways in gas-1(fc21) adult worms

Transcriptome expression changes in the KEGG biochemical pathway that were significantly upregulated in gas-1(fc21) relative to wild-type (N2 Bristol) worms (first comparison, p<0.05) were evaluated in gas-1(fc21) mitochondrial complex I mutant nematodes treated for 24 hours during early adulthood with one of (A) six water-soluble drugs relative to S. basal control or (B) lipophilic vitamin E relative to ethanol control. Wild-type worms (N2 Bristol) were treated only with buffer control. Each bar indicates comparative pathway changes in second group listed as compared to first. Enrichment score conveys degree to which upregulated KEGG biochemical pathways in gas-1(fc21) relative to N2 control were normalized by each treatment in gas(fc21) worms.

Figure 6. Antioxidant drug effects on downregulated KEGG pathways in gas-1(fc21) adult worms

Transcriptome expression changes in the KEGG biochemical pathway that were significantly downregulated in gas-1(fc21) relative to wild-type (N2 Bristol) worms (first comparison, p<0.05) were evaluated in gas-1(fc21) mitochondrial complex I mutant nematodes treated for 24 hours during early adulthood with one of (A) six water-soluble drugs relative to S. basal control or (B) lipophilic vitamin E relative to ethanol control. Wild-type worms (N2 Bristol) were treated only with buffer control. Each bar indicates comparative pathway changes in second group listed as compared to first. Enrichment score conveys degree to which upregulated KEGG biochemical pathways in gas-1(fc21) relative to N2 control were normalized by each treatment in gas-1(fc21) worms.

Figure 7. N-acetylcysteine rescues the decreased cellular viability and increased mitochondrial oxidant burden that occur in RC complex I-deficient human fibroblasts from Leigh syndrome subjects

(A) ND5 (m.13513G>A) heteroplasmic mutant complex I deficient human fibroblasts (Q1269 line) have greater than 95% death when grown for 5 days in glucose-free media, whereas the control line from her mother who does not carry the mtDNA pathogenic mutation in ND5 has 43% death under the same growth conditions. p < 0.001, n=3 biological replicates. (B) Remarkably, NAC [1 mM] co-treatment fully restored viability in the ND5 line grown in glucose-free conditions for 5 days. (C) NDUFS8 compound heterozygous mutant (c.160C>G;P.R54W; c.58G>C:p.G20R) complex I deficient human fibroblasts (Q1508 line) have increased mitochondrial oxidant burden relative to either heterozygous, unaffected parental control fibroblast line (Q1508p1 and Q1508p2). Relative fluorescence quantitation of Mitotracker Green (MTG) and MitoSOX (SOX) were measured by fluorescence-activated cell sorting (FACS) analysis. n=3 per condition. *, p<0.05. (D) NDUFS8 compound heterozygous mutant cells had reduced mitochondrial oxidant burden when treated with 100 μM NAC. Cells were cultured in 10% FBS DMEM media containing 5.6 mM glucose for 24 hour, p<0.05s.

Figure 8. N-Acetylcysteine (NAC) and Vitamin E treatments prevented rotenone-induced cell death in zebrafish larvae brain

(A) Control (EtOH/DMSO) larvae at 7 dpf have normal brain morphology, development, and swimming behavior. (B) Larval treatment with rotenone (30–100 nM) for 4 hours on 7 dpf causes cellular death in their brains as evidenced by grey tissue (asterisk). (C) Pre-treatment with NAC prevented brain necrosis. (D) While acute rotenone exposure on 7 dpf induced brain death in 83% of buffer-only treated larvae (n=53), brain death was significantly reduced to 49% of animals who received pre-treatment from 3.5 dpf with 1 mM NAC (n=55, p=0.006). (E) Vitamin E (25 μM) pre-treatment from 6 dpf led to near-complete rescue of brain death when zebrafish larvae were later exposed to rotenone on 7 dpf (p < 0.001). **, p < 0.01; ***, p < 0.001.