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Considerable evidence suggests that mitochondrial dysfunction and oxidative stress contribute to the progression of Alzheimer's disease (AD). We examined the ability of the novel mitochondria-targeted antioxidant MitoQ (mitoquinone mesylate: [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cycloheexadienl-yl) decyl triphenylphosphonium methanesulfonate]) to prevent AD-like pathology in mouse cortical neurons in cell culture and in a triple transgenic mouse model of AD (3xTg-AD). MitoQ attenuated β-amyloid (Aβ)-induced neurotoxicity in cortical neurons and also prevented increased production of reactive species and loss of mitochondrial membrane potential (Δψ(m)) in them. To determine whether the mitochondrial protection conferred by MitoQ was sufficient to prevent the emergence of AD-like neuropathology in vivo, we treated young female 3xTg-AD mice with MitoQ for 5 months and analyzed the effect on the progression of AD-like pathologies. Our results show that MitoQ prevented cognitive decline in these mice as well as oxidative stress, Aβ accumulation, astrogliosis, synaptic loss, and caspase activation in their brains. The work presented herein suggests a central role for mitochondria in neurodegeneration and provides evidence supporting the use of mitochondria-targeted therapeutics in diseases involving oxidative stress and metabolic failure, namely AD.
MitoQ inhibited Aβ-induced death of C57BL/6 mouse cortical neurons in cell culture.
A
, Photomicrographs of untreated cortical neurons and cortical neurons exposed to Aβ
(22–35)
(25 μ
m
) alone or with MitoQ for 48 h. This concentration was chosen because similar concentrations of truncated or full-length Aβ peptides are reported to be toxic to cells in culture (Mazziotti and Perlmutter, 1998).
B
, MitoQ prevented most cell death caused by Aβ
(22–35)
.
n
= 30 cultures per condition except for 100 n
m
MitoQ or dTPP in which
n
= 9 cultures per condition.
C
, MitoQ also prevented Aβ
(1–40)
-induced death.
n
= 9 cultures per condition. After treatments, neurons were fixed, counted, and quantified as percentage of the average number of healthy (phase-bright) neurons in control, sibling cultures. Identical letters in this and subsequent figures indicate conditions that are not statistically different (
p
> 0.01). Different letters in this and subsequent figures signify that the conditions are significantly different from conditions with other letters (
p
< 0.01).
MitoQ prevented increased Aβ-induced RS production and Aβ-induced Δψ
m
depolarization in C57BL/6 mouse cortical neurons in cell culture. Confocal microscopy allowed simultaneous assessment of RS by the redox-sensitive dye CM-H
2
DCFDA and Δψ
m
by the potential-dependent fluorescent dye TMRM
+
.
A
, Confocal micrographs of mouse cortical neurons costained with CM-H
2
DCFDA (green) and TMRM
+
(red). RS levels, indicated by increased CM-H
2
DCFDA intensity, were much higher in cells treated for 24 h with Aβ
(22–35)
(25 μ
m
) peptide. MitoQ (1 n
m
) blocked the increased RS caused by exposure of neurons to Aβ
(22–35)
.
B
, MitoQ and the NOS inhibitor
l
-NNA suppressed increased RS caused by Aβ
(22–35)
exposure. dTPP, which mimics the mitochondria targeting moiety of MitoQ but contains no ubiquinone, had no significant effect. The uncoupling agent FCCP blocked RS at a concentration that eliminates Δψ
m
in neurons (Kirkland et al., 2010).
C
, MitoQ and
l
-NNA also blocked depolarization of Δψ
m
(decreased TMRM
+
intensity) caused by Aβ
(22–35)
peptide, whereas dTPP had no effect. Dye intensity was determined in confocal micrographs with MetaMorph software and normalized to the average dye intensity of control cultures plated at the same time. Dye intensities are shown as fold change from these values.
MitoQ treatment prevented the onset of cognitive deficits in young female 3xTg-AD mice.
A
, Time courses of spatial learning and memory acquisition in the MWM. At 6.5 months of age, five groups of female mice having the specified genotypes and that had received the indicated treatments for 4.5 months were trained in the MWM (for details, see Materials and Methods). Each mouse performed four training trials per day for 7 consecutive days, and the latencies in seconds for each animal to reach a platform hidden under opaque water per day were averaged. All trials were filmed by digital camera, and the data were analyzed by EthoVision software. All groups were able to perform the task with equal latencies after 7 d of training. However, the nonTg, nonTg + MitoQ, and 3xTg-AD + MitoQ mice learned the task more quickly than did untreated or dTPP-treated 3xTg-AD mice (
p
< 0.01 by longitudinal regression analysis, indicated by asterisks). Treated mice received either dTPP (100 μ
m
) or MitoQ (100 μ
m
) in their drinking water from 2 to 7 months after birth.
B
, MitoQ treatment prevented loss of short- and long-term spatial memory retention in the 3xTg-AD mice. Memory retention was assessed in MWM probe trials conducted 1.5 and 24 h after the last training trials. In the probe trials, the platform was removed from the water tank, and mice were allowed a 60 s free swim. Spatial bias is shown as the number of previous platform location crosses as determined by video analysis using Ethovision tracking software. MitoQ significantly prevented both short- and long-term retention deficits in 3xTg-AD mice. Conversely, dTPP treatment did not significantly affect learning or memory in these mice.
C
, Cued acquisitions trials in which the platform was not hidden revealed that all mice were capable of swimming to the visible platform in the same amount of time (
p
= 0.7 by ANOVA).
n
= 21–38 mice except for the MitoQ-exposed nonTg in which 12 animals were treated.
MitoQ prevented oxidative stress in the brains of young female 3xTg-AD mice.
A
, MitoQ treatment inhibited decreased GSH/GSSG ratios in the brains of 3xTg-AD mice (
p
< 0.01). As a positive control, nonTg samples were incubated with 40 μ
m
of the O
2
·−
and, by dismutation, H
2
O
2
generator menadione (MND) for 45–60 min before the assay to oxidize GSH to GSSG (Di Monte et al., 1984). The results are expressed as fold decrease of GSH/GSSH from control level in nonTg brains.
n
= 6–10 brains.
B
, MitoQ treatment prevented increased lipid peroxidation (MDA levels) in the brains of 3xTg-AD mice (
p
< 0.01). MDA levels were determined by the TBARS assay.
n
= 6 brains for each except dTPP in which
n
= 3 brains.
C
, MitoQ treatment suppressed the development of elevated levels of 3-NT in the brains of 3xTg-AD mice. Top, Representative immunoblots showing that 3-NT levels were higher in two proteins in the brains of 3xTg-AD mice than they were in the same proteins in nonTg mice. 3-NT levels in these proteins were lower in the brains of MitoQ-treated 3xTg-AD animals than in untreated or dTPP-treated ones. Bottom, Quantitative analysis of 3-NT band density indicated that nitrated proteins were significantly increased in untreated 3xTg-AD mice at 7 months of age and that this increase was prevented by MitoQ treatment. Density was determined for entire lanes and is shown as fold change from the average density in nonTg brains.
n
= 5–6 brains. The brains used in this and subsequent assays were obtained from the 7-month-old female mice (Fig. 3) that had been treated as indicated for 5 months. Brains were removed 2 weeks after the end of MWM probe trials.
MitoQ prevented synaptic loss and proliferation of reactive astrocytes in the brains of young female 3xTg-AD mice.
A
, Immunoblots demonstrating that MitoQ prevented loss of the synaptic protein synaptophysin (Syn) and increase of the astrocyte marker GFAP in the brains of 3xTg-AD mice. Blots between lines are all from the same gel. Similar amounts of protein, as determined by the Bradford assay, were loaded onto the gels. Decreases in synaptophysin (
B
) and increases in GFAP (
C
) in the brains of 3xTg-AD mice as normalized to the levels in wild-type mice were blocked by MitoQ treatment.
n
= 4–9 brains for synaptophysin and 3–8 brains for GFAP.
MitoQ reduced Aβ
(1–42)
burden in brains of young 3xTg-AD mice.
A
, Representative photomicrographs showing immunostaining with an anti-Aβ
(1–42)
antibody within the hippocampus (top) and neocortex (bottom) of nonTg, untreated 3xTg-AD mice and 3xTg-AD mice that had received MitoQ treatment for 5 months. Many cells in 3xTg-AD mice receiving no treatment exhibited cytosolic staining, whereas 3xTg-AD mice that had received MitoQ treatment exhibited much less staining. Sections are representative of four brains per condition.
B
, Quantification of soluble Aβ
(1–42)
by ELISA revealed that MitoQ treatment prevented increased Aβ
(1–42)
burden in the 3xTg-AD mouse brain.
n
= 4 brains for each condition.
MitoQ treatment blocked increased caspase 3/7 activity in young female 3xTg-AD mouse brains. Caspase 3/7 activity was assessed by the Caspase-Glo 3/7 assay. Brain homogenates were incubated with a proluminescent caspase 3/7-specific substrate and luminescence quantified by a luminometer. More intense luminescence indicates higher caspase 3/7 activity. Values are normalized to the average luminescence of the nonTg controls.
n
= 8–10 brains.
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MC_U105663142/MRC_/Medical Research Council/United Kingdom
R01 NS037110/NS/NINDS NIH HHS/United States
R29 NS037110/NS/NINDS NIH HHS/United States
NS37110/NS/NINDS NIH HHS/United States
