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Oxidative damage and mitochondrial dysfunction are implicated in aging and age-related neurodegenerative diseases, including Huntington’s disease (HD). Many naturally occurring antioxidants have been tested to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and the efficacy is marginal in human clinical trials. To increase specificity and efficacy, we have designed a synthetic antioxidant, XJB-5-131, to target mitochondria. We demonstrate in a mouse model of HD that XJB-5-131 has remarkably beneficial effects. XJB-5-131 reduces oxidative damage to mitochondrial DNA, maintains mitochondrial DNA copy number, suppresses motor decline and weight loss, enhances neuronal survival, and improves mitochondrial function. The findings poise XJB-5-131 as a promising therapeutic compound.
Defective mitochondria (MT) are implicated in the pathogenesis of neurodegenerative diseases including Alzheimer’s disease (AD) (Huang and Mucke, 2012), Parkinson’s disease (PD) (Exner et al., 2012), and Huntington’s disease (HD) (Johri and Beal, 2011). In all of these disorders, accumulation of oxidative damage is involved (Exner et al., 2012; Huang and Mucke, 2012; Johri et al., 2011), but there are no effective therapeutics (Hersch and Rosas, 2008; Mangialasche et al., 2010). MT are the primary intracellular source of reactive oxygen species (ROS) and the main target for oxidative damage (Lin and Beal, 2006). Thus, dietary supplements of naturally occurring antioxidants such as vitamin E and coenzyme Q10 (CoQ10) have been used in mice and in human clinical trials to alleviate the deleterious effects of MT-dependent ROS production (Dumont et al., 2011; Littarru and Tiano, 2010). In general, however, their efficacy has been marginal or tissue variable (Kwong et al., 2002; Sohal and Forster, 2007; Sumien et al., 2009), and it remains controversial whether dietary supplementation of CoQ10 can increase its steady-state level in the brain in vivo (Kwong et al., 2002; Sohal and Forster, 2007). These observations have driven efforts to enhance specificity and therapeutic efficacy by direct targeting of synthetic antioxidants to MT.
XJB-5-131 is a bi-functional antioxidant that comprises a radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl nitroxide (Figure 1A, red) conjugated to a mitochondrial targeting moiety. The targeting portion of the molecule is an alkene peptide isostere modification of the Leu-D-Phe-Pro-Val-Orn segment of the antibiotic, gramicidin S (Hoye et al., 2008; Wipf et al., 2005) (Figure 1A, black) that localizes to the mitochondrial membrane. Previously, we have reported that XJB-5-131 reduces apoptosis and enhances cell survival in mouse embryonic cells in vitro (Wipf et al., 2005). In this work, we tested the beneficial effects of XJB-5-131 in a mouse model of HD. We find that XJB-5-131 significantly suppresses the disease phenotypes and improves mitochondrial function.
We evaluated the efficacy of XJB-5-131 in a mouse model of HD harboring disease-length 150 CAG tract “knocked-into” both alleles of the mouse HD gene homologue (Hdh(CAG)150/(CAG)150) (Lin et al., 2001). These animals will be referred to hereafter as HD150KI. To validate that XJB-5-131 targeted MT, we isolated primary striatal neurons (embryonic day 17) from HD150KI animals and treated them with BODIPY-FL-XJB-5-131, a derivative of XJB-5-131 labeled with the fluorescent (FL) boron-dipyrromethene (BODIPY) (Figure 1A). Within one hour of incubation, BODIPY-FL-XJB-5-131 crossed the plasma membrane and stained MT in primary neurons, as verified by co-staining with MitoTracker Deep Red (Figure 1B). Treatment of freshly plated primary neurons with XJB-5-131 (1 µM) for 7 days did not induce measureable changes in the number of MT (Figure 1C). However, under these conditions, XJB-5-131 improved survival of the primary striatal neuronal cultures derived from both HD150KI mice (Figure 1D) and genetically matched C57BL/6 wild type mice (Figure S1), suggesting that accumulation of XJB-5-131 in the MT was beneficial to the primary neuronal cultures in vitro.
We tested whether XJB-5-131 had beneficial effects in vivo. We intraperitoneally injected HD150KI mice with XJB-5-131 at 1 mg/kg three times a week up to 57 weeks, and tested whether treatment improved the disease phenotypes in these animals. Chronic treatment of HD150KI mice with XJB-5-131 suppressed weight loss, a pathology commonly observed in HD patients (Kirkwood et al., 2001). Genetically matched C57BL/6 control animals increased in weight to an average of 44 ± 5 g at 57 weeks (Figure 2A), while age- and gender-matched HD150KI mice were smaller (32 ± 3 g) (Figure 2A). Treatment of HD150KI animals with XJB-5-131 increased the average body mass by 22% (Figure 2A).
Early signs of HD pathology manifest as motor abnormalities (Kirkwood et al., 2001). As a second test for XJB-5-131’s efficacy, we measured rotarod performance and grip strength of animals at 9, 28, and 57 weeks (Figure 2B). All animals at 9 weeks performed equally well on the rotarod (Figure 2B). However, performance in HD150KI mice declined with age, resulting in an approximately 70% decrease by 57 weeks (Figure 2B). Remarkably, there was no significant decline in rotarod performance in HD150KI mice chronically treated with XJB-5-131 during the same period (Figure 2B). Furthermore, XJB-5-131 treatment led to a striking improvement in grip strength (Figure 2C). At 57 weeks, approximately 95% of the HD150KI mice failed to grip the bar for 30 seconds in three trials, while at the same age, 85% of the XJB-5-131-treated HD150KI littermates passed the test. Collectively, these data demonstrated that XJB-5-131 significantly reduced the disease phenotypes observed in HD150KI mice.
MT are the primary intracellular source of ROS and the main target for oxidative damage (Lin and Beal, 2006; Woo and Shadel, 2011). If the beneficial effects of XJB-5-131 in MT were due to its antioxidant properties in vivo, then we anticipated that a reduction in oxidative damage to mtDNA would accompany the improvement in motor function. To test the hypothesis, we applied quantitative PCR (qPCR) to measure whether XJB-5-131 altered the level of oxidative damage to mtDNA. Because the movement of the polymerase on the template is blocked at a lesion, qPCR amplification is inversely proportional to the presence of DNA damage. HD150KI animals had a remarkable increase in the load of oxidative mtDNA damage relative to age-matched controls (Figures 3A and 3C). However, treatment with XJB-5-131 led to a reduction in the damage burden (Figures 3A and 3C). Similarly, the abundance of mtDNA molecules in HD150KI animals decreased by 36% relative to controls (Figures 3B and 3D), consistent with previous suggestions that mitochondrial biogenesis is impaired in HD animals (Johri et al., 2012). Chronic treatment with XJB-5-131 restored the mtDNA abundance to control levels (Figures 3B and 3D). Thus, XJB-5-131 treatment not only decreased the lesion load in mtDNA but also prevented a decline in the relative abundance of mtDNA in treated HD150KI animals relative to untreated littermates (Figures 3A-3D).
Because XJB-5-131 is a MT-targeted antioxidant, we tested its effects on mitochondrial function. MT generate most of the cell's supply of ATP via the oxidative phosphorylation pathway, which is measured by the oxygen consumption rate (OCR) (Zhang et al., 2012). To determine the effects of XJB-5-131 on OCR, we isolated synaptosomes from 57-week HD150KI animals and compared OCR before and after treatment with inhibitors of the electron transport chain (Figure 4A). Synaptosomes are “pinched off” nerve terminals that harbor intact neuronal MT and represent a simple and robust system to assess mitochondrial function within a physiological milieu (Choi et al., 2009). Treatment of synaptosomes with 200 nM XJB-5-131 did not induce a measureable change in the relative OCR of resting synaptosomes from HD150KI animals (Figure 4B). Similarly, XJB-5-131 had little effect on the relative decrease in OCR in HD150KI synaptosomes after inhibition of ATP synthase with oligomycin (Figure 4B). These results implied that XJB-5-131 had minimal effects on mitochondrial metabolism in the resting state. However, XJB-5-131 significantly improved mitochondrial response to cellular stress as measured by the mitochondrial membrane potential uncoupler, fluoro-carbonyl cyanide phenylhydrazone (FCCP) (Figure 4B). FCCP destroys the proton gradient in MT (Figure 4A) and uncouples electron transport from ATP generation. Under these conditions, OCR rises as the MT try to “keep up” with the energy demands of the cell. The rise in OCR after FCCP treatment reflects the maximum ability of MT to maintain energy production in response to acute and chronic stress (Dranka et al., 2010), and is referred to as mitochondrial spare respiratory capacity (SRC) or reserve capacity (Figure S2). Indeed, treatment of striatal synaptosomes with 1 µM and 10 µM solutions of XJB-5-131 elevated the mitochondrial SRC, as measured by the approximately 2-fold increase in OCR relative to untreated striatal synaptosomes (Figure 4C). XJB-5-131 treatment conferred a similar increase (2-3 fold) on the mitochondrial SRC in HD150KI cortical synaptosomes (Figure S2) and in striatal synaptosomes isolated from genetically matched C57BL/6 wild type mice (Figure S2), indicating that the improvement in the mitochondrial stress response was neither limited to the striatum nor specific for the HD mouse model.
XJB-5-131 was designed as a targeted antioxidant. Thus, we tested whether the protective effect of XJB-5-131 against ROS-induced damage was due, at least in part, to the radical scavenging activity of the compound. The ROS-inducer, DMNQ is a non-thiol-capturing and non-alkylating redox cycling quinone that causes continuous intracellular generation of H2O2 and subsequent oxidative damage to MT (Figure 4D) (Parry et al., 2009). If XJB-5-131 was a radical scavenger, then we anticipated that it would protect against DMNQ-induced mitochondrial damage and would alter the OCR in synaptosomes in response to DMNQ exposure. As expected, treatment with oligomycin and FCCP increased OCR in synaptosomes from 57-week HD150KI animals (Figure 4E). Addition of DMNQ, under these conditions, reduced the SRC (Figure 4E), while administration of XJB-5-131 and DMNQ together recovered the mitochondrial SRC to 90% of control (Figure 4F). The XJB-5-131–induced increase in mitochondrial SRC was robust since subsequent addition of rotenone and antinomycin A, inhibitors of complex I and complex III (Figure 4E), respectively, eliminated OCR differences among treatment groups. These results suggested that the beneficial effects of XJB-5-131 were due, at least in part, to its radical scavenging activity, as designed.
Generic and naturally occurring antioxidants have been largely ineffective against neurodegenerative diseases, although mitochondrial dysfunction has been strongly implicated as part of the toxic mechanism (Johri and Beal, 2011; Mangialasche et al., 2010). Here we demonstrate that a synthetic, MT-targeted XJB-5-131 imparts a remarkable suppression of motor decline, inhibits weight loss, reduces mtDNA damage, maintains mtDNA copy number, improves mitochondrial function, and enhances neuronal survival in a knock-in mouse model of HD. Collectively, these findings imply that specific targeting of this synthetic antioxidant to MT has beneficial effects both in vitro and in vivo, and poise XJB-5-131 as a promising therapeutic compound.
XJB-5-131 has attractive properties. First, its delivery to MT does not depend on the potential gradient across the inner mitochondrial membrane. Rather, selective localization of XJB-5-131 is accomplished via insertion of the β-turn motif within the Leu-D-Phe-Pro-Val-Orn segment into the mitochondrial membrane (Fink et al., 2007). Thus, compromised MT with reduced membrane potential would not exclude entry of XJB-5-131. Indeed, we demonstrate that XJB-5-131 significantly improves mitochondrial function upon the treatment with the membrane potential uncoupler FCCP. This is in contrast to potential-dependent delivery of MT-targeted antioxidants. For example, MitoQ and MitoVit E protect against glutathione depletion in cultured fibroblasts from Friedrich’s ataxia patients, yet their enhanced potency is abolished in cells pretreated with FCCP (Jauslin et al., 2003). Moreover, the uptake of these potential-dependent antioxidants is self-limiting, as there is inevitable depolarization of MT upon accumulation of large amounts of cations in the matrix (Kelso et al., 2001; Smith et al., 1999). Second, XJB-5-131 readily crosses the plasma membrane while exogenously supplemented vitamin E and CoQ10 are more lipophilic and tend to be retained in cell membranes. Thus, these naturally occurring compounds have difficulty in achieving pharmacologically significant intracellular concentrations (Jauslin et al., 2003; Szeto, 2006).
XJB-5-131 not only enhances mitochondrial SRC but also protects against DMNQ-induced loss of mitochondrial SRC. Mitochondrial SRC is critical for survival and function of cells, especially under conditions where cells undergo increased oxidative stress or a sudden increase in energy demand (Desler et al., 2012). For example, enhanced mitochondrial SRC favors T cell survival after infection (van der Windt et al., 2012) and loss of mitochondrial SRC renders cell death in the rotenone model of PD with partial Complex I deficiency (Yadava and Nicholls, 2007). In our experiments, the favorable properties of MT-targeted XJB-5-131 suppressed pathophysiology without obvious toxicity. Collectively, the beneficial effects of XJB-5-11 are promising, and warrant further investigations for its efficacy in a broader set of MT-associated disorders and premature aging phenotypes.
More detailed experimental procedures are provided in the Supplemental Information.
We used a mouse model of HD (Lin et al., 2001), referred to as HD150KI mice. Genetically matched C57BL/6 mice were used as controls unless otherwise stated. All procedures involving animals were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Protocols were approved by the Lawrence Berkeley National Laboratories Animal Welfare and Research Committee.
XJB-5-131 was synthesized as described previously (Wipf et al., 2005). A Fluorescent (FL) boron-dipyrromethene (BODIPY)-labeled XJB-5-131 was prepared in an analogous fashion. HD150KI mice were intraperitoneally injected 1 mg/kg of XJB-5-131 or phosphate buffered saline three times per week from 7 to 57 weeks. Animals in each group (10 for XJB-5-131 treatment group and 7 for untreatment group) were evaluated for rotarod performance and grip strength at 9, 28, and 57 weeks on a Rota-Rod apparatus (Ugo Basile, Italy) according to a protocol described previously_ENREF_3 (Trushina et al., 2006). See Supplemental Information for details.
Striatal neurons isolated on embryonic day 17 from HD150KI mice were cultivated as described elsewhere (Trushina et al., 2004). MitoTracker Deep Red (Invitrogen, Carlsbad, CA) and BODIPY-FL-XJB-5-131 were used to label MT in live primary striatal neurons. Images were acquired on a Zeiss LSM 710 inverted laser scanning confocal microscope. Image J and FIJI was used for image analysis and quantification of cells. See Supplemental Information for details.
The amplification of a 10 kb and a 116 bp mtDNA fragment was used to detect lesions and abundance in mouse cerebral cortex as previously described (Ayala-Torres et al., 2000; Santos et al., 2006). See Supplemental Information for details.
Striatal and cortical synaptosomes were isolated as previously described (Choi et al., 2009). Protein concentration was determined using the Bio-Rad Bradford assay (BioRad Laboratories). See Supplemental Information for details.
Respiration of synaptic MT was measured by the oxygen consumption rate (OCR) using a Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA) according to the manufacturer's instructions. See Supplemental Information for details.
Freshly isolated synaptosomes were exposed to 1 µM DMNQ either in the absence or presence of 10 µM XJB-5-131. Synaptosomes were plated on the Seahorse PS 96-well microplate and spun down for 50 minutes at 4°C. The DMNQ exposure time was approximately 90 minutes before the OCR was measured.
Values were expressed as mean ± standard error of the mean (SEM), unless otherwise stated. P-values were obtained from the unpaired two-tailed Student's t-test.
This work was supported by the National Institutes of Health grants NS40738 (to CTM), GM066359 (to CTM), NS062384 (to CTM), NS060115 (to CTM), CA092584 (to CTM), GM067082 (to PW), AI068021 (to PW), AG024827 (to PDR), AR051456 (to PDR), ES016114 (to LJN), CA103730 (to LJN), the Ellison Medical Foundation (AG-NS-0303-05) (to LJN), and the University of Puerto Rico infrastructural grant (2G12-RR003051) (to EFR).
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CONFLICT OF INTEREST
The authors have declared that no conflict of interest exists.