Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington's disease

Abstract

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 for their ability to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and have marginal efficacy 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.

Figure 1. XJB-5-131 is localized to mitochondria (MT) and enhances neuronal survival in a mouse model of HD

(A) Schematic diagram of the structure and functional design of XJB-5-131 and BODIPY-fluorescent labeled (FL)-XJB-5-131. The structure in red is a stable nitroxide free-radical with reactive oxygen species (ROS) scavenging abilities. The structure in black is derived from a cyclopeptide antibiotic, gramicidin S, which selectively accumulates within microbial cell membranes. BODIPY-FL-XJB-5-131 was designed to validate its mitochondrial localization. (B) MitoTracker Deep Red (MtT, red) and BODIPY-FL-XJB-5-131 (XJB, cyan) co-label MT in primary striatal neurons from embryonic day 17 HD150KI mice. The arrowheads indicate representative MT detected by both probes. Scale bar is 10 microns. (C) XJB-5-131 treatment (1 µM) for a week does not induce measureable changes in the number of MT in primary striatal neurons as quantified after staining with MitoTracker Deep Red intensity. The right panels are representative images. The total number of cells analyzed per condition is >10,000 from three independent experiments. Data are mean ± SEM (n = 3). Scale bar is 10 microns. (D) XJB-5-131 treatment protects survival of primary striatal neurons after 7 days in culture. The right panels are representative images. Scale bar is 10 microns. Data are mean ± SEM (n = 3). **, P < 0.01 using the unpaired two-tailed Student's t-test. XJB-5-131 is abbreviated as XJB in the figures. See also Figure S1.

Figure 2. XJB-5-131 suppresses decline of weight loss and motor function in a mouse model of HD

(A) Weight of 57-week C57BL/6 wild-type (WT, n = 15), untreated HD150KI (− XJB, n= 7), and XJB-5-131-treated HD150KI (+ XJB, n = 10) mice. n is the number of mice used. Data are mean ± SEM, n = 7-15 mice/group. **, P < 0.01 using the unpaired two-tailed Student's t-test. (B) Rotarod performance and (C) grip strength test of animals at the age of 9, 28, and 57 weeks (Wks) with (+ XJB, n = 10) and without (−XJB, n = 7) XJB-5-131 treatment. Grasp test was deemed a pass if the animals held the bar for 30 seconds (within three trials). Each age group of animals was tested together and the results were expressed as % pass. n is the number of mice used. Data aremean ± SEM, n = 7-10. **, P < 0.01 using the unpaired two-tailed Student's t-test. XJB-5-131 is abbreviated as XJB in the figures.

Figure 3. XJB-5-131 reduces mtDNA damage and maintains mtDNA copy number

(A) Representative agarose gel of the amplification of a 10 kb mtDNA fragment from WT, HD150KI, HD150KI + XJB-5-131 treated mice. (B) Representative agarose gel of the amplification of a 116 bp mtDNA fragment from WT, HD150KI, HD150KI + XJB-5-131 treated mice. (C) XJB-5-131 decreases the levels of mtDNA lesions in the cerebral cortex of HD150KI mice. Results were derived from three qPCR assays performed in duplicate on each animal. The number of mice used was 6, 7, and 6 for control, HD150KI, and HD150KI + XJB-5-131, respectively. Data are mean ± SEM (n = 6–7 mice/group). **, P < 0.01 using the unpaired two-tailed Student's t-test. (D) XJB-5-131 restores the abundance of mtDNA molecules in cerebral cortex of HD150KI mice. Results were derived from two qPCR assays performed in duplicate on each animal. The number of mice used was 6, 7, and 6 for control, HD150KI, and HD150KI + XJB-5-131, respectively. Data are mean ± SEM (n = 6–7 mice/group). **, P < 0.01 using the unpaired two-tailed Student's t-test. XJB-5-131 is abbreviated as XJB in the figures.

Figure 4. XJB-5-131 improves mitochondrial function in HD150KI animals

(A) Simplified diagram of electron transport chain and its inhibitors. Rotenone, antimycin A, and oligomycin are the inhibitors of Complex I, Complex III, and ATP synthase, respectively. Fluoro-carbonyl cyanide phenylhydrazone (FCCP) is an ionophore, which allows re-entry of the protons into the mitochondrial matrix and dissipates the proton gradient. (B) Representative profiles showing effects of XJB-5-131 treatment on oxygen consumption rates (OCR) under basal conditions and upon injections of mitochondrial inhibitors in isolated striatal synaptosomes from 57-week HD150KI mice. Each profile represents one independent biological experiment analyzed in triplicate. Data are means ± SEM (n = 3). The arrows indicate the injection of mitochondrial inhibitors. Three independent experiments were performed to obtain quantification of mitochondrial spare respiratory capacity (SRC) presented in (C). (C) Effects of XJB-5-131 treatment on SRC in isolated striatal synaptosomes. SRC is defined as the OCR difference between FCCP-induced respiration and basal respiration. Data represent three independent experiments and each was performed with five HD150KI mice and analyzed in triplicate. Data are mean ± SEM (n = 3). *P < 0.05, **, P < 0.01 using the unpaired two-tailed Student's t-test. (D) Schematic diagram illustrating the working hypothesis of DMNQ and XJB-5-131 within MT. (E) Representative profiles showing effects of DMNQ on OCR in the absence and presence of XJB-5-131 under basal conditions and upon injection of mitochondrial inhibitors. The down arrows indicate the injection of mitochondrial inhibitors. Each profile represents one independent biological experiment analyzed in triplicate. Data are means ± SEM (n = 3). Three independent experiments were performed to obtain quantification of mitochondrial SRC presented in (F). (F) XJB-5-131 treatment significantly improves mitochondrial SRC in 57-week HD150KI mouse striatal synaptosomes resulting from 1 µM DMNQ exposure. Data are normalized to the control (in the absence of both XJB-5-131 and DMNQ) and are representative of three independent experiments. In each experiment, three HD150KI mice were used and analyzed in triplicate. Data are mean ± SEM (n = 3). *, P < 0.05 using the unpaired two-tailed Student's t-test. XJB-5-131 is abbreviated as XJB in the figures. See also Figure S2.