Mech Ageing Dev. 2012 (Aug); 133 (8): 523–529 ~ FULL TEXT
Jiangang Long, Vadim Aksenov. David Rollo, Jiankang Liu
Institute of Mitochondrial Biology and Medicine,
Department of Biology and Engineering,
The Key Laboratory of Biomedical Information Engineering of Ministry of Education,
Xi'an Jiaotong University School of Life Science and Technology,
Xi'an 710049, China.
We examined whether transgenic growth hormone mice (Tg) that exhibit accelerated cognitive aging and exceptional free radical damage also express elevated nitrative stress. We characterized age-related patterns of 3-nitrotyrosine (3-NT) in brain homogenate and mitochondria of Tg and normal (Nr) mice as modulated by a complex anti-aging dietary supplement. Levels of 3-NT rose rapidly with age in Tg brain homogenate whereas normal controls maintained constant lower levels. The age-related slope for 3-NT was 3.6-fold steeper in untreated Tg compared to treated Tg (p<0.009), although treated Tg showed elevation in youth. Opposite to Tg, treated Nr mice had reduced 3-NT in youth (p<0.02). The age-related pattern of mitochondrial 3-NT in Nr mice was parabolic (p<0.005). Remarkably, levels in treated Nr were reduced by ~50% (p<0.0007). Untreated Tg showed strongly increasing mitochondrial 3-NT with higher mitochondrial activity (p<0.01) whereas treated Tg showed lower nitrosylation at higher levels of mitochondrial activity. Tg mice also expressed a postural abnormality that is a biomarker of neurodegeneration and/or nitrative stress. Tg represent a promising new model of nitrative stress associated with brain deterioration and results provide proof of principle that complex dietary supplements may be ameliorating.
From the FULL TEXT Article:
We assessed reactive nitrogen species damage in normal (Nr)
and transgenic growth hormone mice (Tg) using 3-nitrotyrosine
(3-NT). Elevated 3-NT is a reliable biomarker of nitrative stress,
aging and associated pathologies (Radi, 2004), particularly
cognitive and somatosensory dysregulation as occurs in Alzheimer’s,
Parkinson’s and Huntington’s diseases (Ansari and Scheff,
2010; Butterfield et al., 2007; Calabrese et al., 2009; Knott and
Bossy-Wetzel, 2009; Steinert et al., 2010). Peroxynitrite generated
via interaction of superoxide and nitric oxide (NO) mainly
contributes to 3-NT formation and also impacts membranes,
catalytic activity, cytoskeletal organization, and cell signaling
pathways (Nuss et al., 2009; Pacher et al., 2007).
Mitochondria are the primarily source of free radicals and
substantially contribute to reactive nitrogen species (Wei, 1998).
Mitochondrial neuronal nitric oxide synthase (mtNOS) generates NO
in immediate proximity to high concentrations of superoxide, thus
favoring formation of peroxynitrite (Finocchietto et al., 2009). A
cytoplasmic nNOS may also contribute to reactive nitrogen species.
While diffusion of NO is rapid and it can react with superoxide even
in adjacent cells (Pacher et al., 2007), highest nitrative damage is
likely to occur near the source. Therefore, we quantified mitochondrial-
derived 3-NT separately from whole-cell homogenate.
Transgenic growth hormone mice (Tg) express elevated free
radical processes in all tissues examined (Rollo et al., 1996) and
display accelerated aging with pronounced declines in cognitive and
motor functions (Aksenov et al., 2010; Lemon et al., 2003, 2005). We
developed a complex dietary supplement (DSP) targeting five key
mechanisms of aging (oxidative stress, inflammation, mitochondrial
function, insulin resistance and membrane integrity). The DSP
strongly ameliorated functional aging and age-associated pathologies
in Tg and Nr mice (Aksenov et al., 2010; Lemon et al., 2003,
2005). Here we confirm Tg as a new model of nitrative stress and
show that the DSP ameliorated nitrative processes in both genotypes
(particularly in mitochondria). This suggests promise for neurodegenerative
conditions where nitrative stress is one likely mechanism
(Kanaan et al., 2008).
Materials and methods
Animals and diets
Breeding and husbandry of random bred C57BL/6J*SJL Nr and Tg mice were
previously described (Aksenov et al., 2010). Protocols adhered to Canada Council on
Animal Care guidelines. Our DSP contained 30 ingredients (Table 1) available
without prescription (Aksenov et al., 2010). Dosages were derived from
recommended human doses adjusted for body size and the 10-fold higher
metabolic rate of mice (Lemon et al., 2005). A thoroughly mixed slurry of the DSP
was soaked onto small pieces of bagel. Mice from the breeding colony were
randomly assigned at weaning and for life to either the DSP treatment group (one
dose/day) or an untreated control group. Bagel bits were avidly eaten ensuring
accurate dosing. Nr and Tg mice in appropriate age ranges were randomly selected
from control and supplemented populations for study.
Preparation of brain homogenate and brain mitochondria
Brains were removed on ice and stored at –80°C. The tissues were homogenized
in 0.05 M PBS with 1 mM 2-mercapitethanol, 1 mM EDTA and 0.1% Trion-x100 as
described (Hinman and Blass, 1981). Brain mitochondria were prepared by
Keeney’s method (Keeney et al., 2006), and protein concentration was determined
with a bicinchoninic acid (BCA) kit.
Mitochondrial complex activities
Mitochondrial complex activities (I–IV) were determined as previously
described (Long et al., 2009a,b). Numbers per group (male mice) were as follows:
untreated Tg, 8; treated Tg, 11; untreated Nr, 10; and treated Nr, 11. Briefly,
complex I activity was assayed by monitoring the decrease of NADH absorption at
340 nm. Final concentration of mitochondria protein was 30 µg/mL. Reaction was
started by adding 200 µmol/L NADH and scanned at 340 nm for 3 min. Rotenone
(3 µmol/L) was added into the reaction system as blank control. Complex II was
assayed in the assay buffer (10x buffer contains 0.5 M phosphate buffer, pH 7.8, 1%
BSA, 10 µM antimycin A, 2 mM NaN3, 0.5 mM coenzyme Q1) with mitochondria
(final concentration 25 µg/mL). The reaction was started with 10 mM succinate and
scanned at 600 nm for 2 min at 30°C. Complex III activity was measured in the
mixture containing 250 mmol/L sucrose, 1 mmol/L EDTA, 50 mmol/L KPi, pH value
adjusted to 6.5 to reduce auto-oxidation of reduced CoQ1, 2 mmol/L KCN, 50 µmol/
L cytochrome C, 0.1% BSA, and the reaction was initiated by 20 µg/mL brain
mitochondria and 50 µmol/L reduced CoQ1, recording the increase of absorption at
550 nm for 2 min. Complex IV was measured by monitoring the decrease of reduced
cytochrome C at 550 nm.
Slot-blot assays of 3-NT and 4-HNE in brain homogenates and mitochondria
3-NT and 4-HNE were measured by slot-blot (Opii et al., 2008; Sultana and
Butterfield, 2008) and relative density was obtained via optical scans. Measurements
were made for both brain homogenates and the mitochondrial fraction. The
3-NT and 4-HNE intensity were represented as relative density units (RDUs).
Tail hang and motor balance tests
For the tail-hang test mice were picked up by the base of the tail and slowly
lowered towards the surface of a bench. Normal mice reach out their forelegs and
splay their legs in anticipation of landing. Mice with neurodegenerative conditions
like the Huntington mouse with high nitrative stress clasp their feet together and
hold the legs close to the body instead of reaching.
Motor function was assessed with the standard Rota-rod test. Mice were placed
on a slowly (5 rpm) rotating cylindrical rod (6 cm in diameter). Latency to fall was
recorded. A soft landing pad 40 cm below cushioned the fall. Each mouse was tested
4 consecutive times. The worst (shortest) performance was discarded and latencies
of the remaining 3 trials were averaged to provide a biomarker of balance. A 2 min
cutoff time was applied.
Global comparisons among all groups were performed using general linear
models. Impacts of diet on Nr and Tg mice were also analyzed separately as
otherwise different trends between genotypes (e.g., opposite patterns and nonlinear
relationships) confounded resolution. Age-related trends were characterized
with linear and polynomial regression. Analysis of covariance (ANCOVA) was
applied to assess impacts of diet when covariates were significant (e.g. age and
mitochondrial complex activity). In all cases separate slopes models were
employed. Descriptive statistics included means ± standard error (SE). Analyses
were performed with Statistica1 software.
Pooled Tg treatments showed an extremely steep age-related
rise in homogenate 3-NT (r = 0.67, p < 0.002, Fig. 1A). This was
particularly accentuated in untreated Tg that showed a regression
slope 3.6-fold steeper than treated Tg (although untreated Tg also
had a lower intercept and lower 3-NT in youth than treated Tg,
Fig. 1A). Linear regression for untreated Tg was highly significant:
3-NT = –23:0872 + 16:6229 (Age), r = 0:84; p < 0:01:
The regression for treated Tg, however, was only marginally
resolved (r = 0.58, p < 0.06) although overall significance was
improved (p < 0.002) when Tg data were pooled. The DSP appeared
to increase 3-NT in young Tg (Fig. 1A). ANCOVA for Tg mice found a
highly significant main effect for the DSP (p < 0.01) and an even
more significant DSP*Age interaction (p < 0.0005). This reflected
the complexity of results (i.e., increases in youthful 3-NT for
treated Tg, but possible amelioration in older ages) (Fig. 1A).
Homogenate 3-NT showed a weak but significant increase with
age in pooled Nr:
Nr 3-NT = 2682:008 + 2:526 (Age), r = 0:45; p < 0:04:
The main effect of supplementation was not resolved (ANCOVA:
p = 0.103) for Nr but a significant DSP*Age interaction emerged
(p < 0.02). Untreated Nr showed stable age-related 3-NT
(slope = 0.491, p > 0.75, Fig. 1B), but treated Nr mice showed
significantly increasing 3-NT with age. Opposite to treated Tg, the
DSP reduced 3-NT in younger Nr mice:
3-NT = 1883:95 + 5:038 (Age), r = 0:71; p < 0:01:
ANCOVA applied to all mice detected highly significant
interaction effects for Genotype*DSP*Age (p < 0.00002) and
Genotype*DSP (p < 0.001) for homogenate 3-NT. Although all Tg
and Nr mice expressed similar levels of homogenate 3-NT in youth
(<150 d), for mice older than 300 d, untreated Tg had 2-fold
greater homogenate 3-NT than untreated Nr mice (p < 0.004, ttest).
The slope for pooled Tg was 3.9-fold greater than pooled Nr
mice and untreated Tg had a slope 6.6-fold steeper than pooled Nr
mice. Overall mean 3-NT for transgenic mice (4951.27 ± 295.04)
was 1.4-fold greater than for normal mice (3587.99 ± 277.43). Both
pooled genotypes showed age-related increases in 3-NT and some
impacts of supplementation. For untreated genotypes, however, Tg
showed remarkably rising 3-NT whereas Nr mice maintained
virtually constant levels of homogenate 3-NT into advanced ages.
Overall, the DSP impacted Tg and Nr homogenate 3-NT oppositely.
Moreover, all aspects were greatly accentuated in Tg mice.
ANCOVA detected a significant genotype effect for mitochondrial
3-NT (p < 0.02) but no main effect of diet. Tg had 1.41-fold
greater levels of mitochondrial 3-NT (6499.82 ± 605.14) than Nr
mice (4616.50 ± 574.08). Pooled Tg data revealed a significant agerelated
decline in mitochondrial 3-NT (p < 0.01, see dichotomy
results below). An impact of diet on Tg mitochondrial 3-NT was
marginally significant (ANCOVA, Age as covariate, p = 0.0509). Both
treated and control Tg showed similar trends for age-related decline
but a significant regression was only resolved for pooled data.
Untreated Nr mice expressed a highly significant parabolic
pattern of mitochondrial 3-NT with age (Fig. 2):
Mitochondrial 3-NT = 1922:187 + 39:724 (Age)
– 0:055 (Age2), r
= 0:86; p < 0:005:
A similar trend in treated Nr mice was not resolved. A simple
comparison for dietary impacts on 3-NT in Nr mice without age as a
covariate was statistically resolved (p < 0.01, t-test). Control Nr
mice had mitochondrial 3-NT levels (6137.7 ± 2968.6) nearly
double those of treated Nr mice (3095.3 ± 2121.7). We also compared
the residuals for normal mitochondrial 3-NT after accounting for the
general parabolic fit (first order polynomial) to the data (i.e., values
obtained by subtracting the observed value from that predicted by the
polynomial regression). This (equivalent to ANCOVA) resolved a
highly significant impact of the supplement on Nr mitochondrial 3-
NT (p < 0.0007).
Levels of mitochondrial lipid peroxidation (4-HNE) were
positively associated with mitochondrial 3-NT in normal mice:
Mitochondrial 3-NT = 1419:61
+ 0:801 Imitochondrial 4-HNE), r
= 0:60; p < 0:003:
This was most pronounced in untreated Nr (Fig. 3):
Mitochondrial 3-NT = 2356:204 + 0:857(HNE), r
= 0:63; p < 0:04:
Treated Nr showed marginal significance (p > 0.06) but
ANCOVA (HNE as covariate) for all Nr mice detected a significant
DSP*HNE interaction (p < 0.01).
Tg showed a similar pattern that was not statistically resolved.
ANOVA detected levels of homogenate 4-HNE in Tg 1.4-fold
higher (4951.27 ± 418.52) than in Nr mice (3587.99 ± 393.99)
(p < 0.02). ANCOVA did not resolve age as a covariate, but rising
levels of 4-HNE were detected in normal homogenate:
Homogenate 4-HNE = 1772:92 + 1:291 (Age), r
= 0:50; p < 0:02:
Mitochondrial complex index
A general index of mitochondrial function (MI) was calculated
by summing activity of complexes I–IV. Exploratory analyses
found that the MI generally resolved clearer patterns and higher
significance than individual complexes alone or an index
calculated by multiplying across individual complexes.
Both treated and untreated Tg showed a trend for declining
homogenate 3-NT with the MI. This was not statistically resolved
using the MI index but pooled Tg were resolved using an index
summing complexes III + IV:
Tg mouse homogenate 3-NT = 9257:66
– 256:199 (complex III + IV), r
= –0:52; p < 0:02
Untreated Nr 3-NT in homogenate showed a significant
negative relationship with the MI, whereas treated Nr showed a
non-significant trend for increase (Fig. 4):
Untreated Nr Homogenate 3-NT = 6505:0 – 57:387 (MI), r
= –0:67; p = 0:048:
Opposite to the homogenate, untreated Tg mitochondrial 3-NT
showed a strong positive association with MI (Fig. 4):
Untreated Tg Mitochondrial 3-NT = –8459:38 + 385:23 (MI), r
= 0:84; p < 0:01:
The relationship appeared exponential, however, and a
logarithmic transformation obtained a fit explaining 86% of the
variance in mitochondrial 3-NT (i.e., r2 = 0.86):Untreated Tg (Log
Mitochondrial 3-NT) = 5.8841 + 0.0695 (MI), r = 0.93, p < 0.001.
A slight negative trend in mitochondrial 3-NT was not resolved
for treated Tg but ANCOVA with the MI as a covariate detected a
strong positive main effect of the DSP (p < 0.002) and a DSP*MI
interaction (p < 0.003). Complex III appeared to particularly
contribute to this pattern but obtained lower statistical resolution
(DSP: p < 0.02; DSP*Mitochondrial Complex III interaction:
p < 0.03).
Nr mitochondrial 3-NT showed no clear relationship to the MI.
However, for any level of MI, mitochondrial 3-NT was ~2-fold
higher in untreated Nr mice (p < 0.01).
Homogenate versus mitochondrial 3-nitrotyrosine dichotomy
Tg expressed opposite age-related patterns of 3-NT in the
mitochondrial compartment compared to homogenate. Homogenate
levels increased by ~4-fold from youth to old age (Fig. 5):
Homogenate 3-NT = 2348:76 + 9:775 (Age), r
= 0:67; p < 0:002:
Alternatively, mitochondrial levels reflected a reciprocal (~5-
fold) decline across the same age range (Fig. 5):
Mitochondrial 3-NT = 9575:81 – 10:857 (Age), r
= –0:54; p < 0:01:
For untreated Tg, mitochondrial 3-NT (6362.8 ± 1312.8) was
1.4-fold higher than in homogenate (4510.8 ± 1106.6). For untreated
Nr mice 3-NT in mitochondria (6137.7 ± 895.1) was 1.7-fold higher
than in homogenate (3689.9 ± 336.3). These patterns highlight the
higher levels of 3-NT in mitochondria and that the greatest
differences in 3-NT between untreated Nr and Tg are in homogenate.
Remarkably, treated Nr mitochondrial 3-NT (3095.3 ± 639.7) was
very similar to treated Nr homogenate 3-NT (3486.1 ± 466.7)(only
11% difference). For treated Tg mitochondrial 3-NT (6636.9 ± 683.4)
was only 1.2-fold greater than 3-NT in homogenate (5391.7 ± 358.0).
Thus, the supplement tended to equalize homogenate and mitochondrial
Behavioral biomarkers of neuropathology
The mouse model for Huntington’s disease (HD) has elevated
nitrosylation in brain associated with a distinct postural abnormality
common to some other neurodegenerative conditions
(Tanaka et al., 2004). When hung by their tail Nr mice reach out
their forelegs towards the surface and splay their hind legs
(Fig. 6A). Huntington mice instead clasp their paws (which can
include the hind feet) (Tanaka et al., 2004). Tg mice hung by their
tails displayed identical paw clasping and leg tucking as found in
the Huntington mouse (Fig. 6B).
In Huntington’s, accumulation of nitrosylated proteins in the
cerebellum results in altered motor function and balance. A
standard test for assessing motor balance involves placing a mouse
on a slowly rotating cylindrical rod (rotarod) (Hamm et al., 1994).
At 5 rpm, this is a mild challenge for Nr mice. Untreated Nr
successfully remained on the rotarod for up to 2 min but Tg fell
within ~30 s (~75% sooner than Nr controls (t-test: p < 0.00001,
diseases. This may be
particularly true for HD. Whereas Nr mice engage a reaching
response when hung by their tail (Fig. 6A). HD mice express the
same paw clasping behavior in the tail hang test as do Tg (Fig. 6B)
(Komatsu et al., 2006). HD mice are a model of nitrative stress and
dysregulated motor balance (Tanaka et al., 2004). Tg similarly
performed very poorly on the rotarod (Fig. 6C). Although this is
considered a standard assay, caution is required as the exceptional
size of Tg may also influence their coordination on this task and Tg
expressed no other obvious symptoms of HD.
Mitochondrial complex index
The index, summing activity of complexes I–IV, is believed
better representing the activity of mitochondrial electron transport
chain than the individual complex activity. It resolved clearer
patterns and higher significance than individual complexes alone
in present study. Mitochondrial 3-NT (Mt 3-NT) increased
exponentially with increasing MI in untreated Tg (Fig. 4).
Supplementation abolished the strong coupling of 3-NT production
to the MI, although it resulted in higher levels of 3-NT
associated with low mitochondrial activity (Fig. 4). ANCOVA
detected significant DSP (p < 0.002) and DSP*MI effects
(p < 0.003). Complex III appeared to particularly contribute to
this pattern as we previously reported for protein carbonyls
(Aksenov et al., 2010).
Nr Mt 3-NT showed no relationship to the MI in terms of slope.
However, for any level of MI, mitochondrial 3-NT was ~2-fold
higher in untreated Nr mice (i.e., parallel flat lines differed in
intercepts). The fact that MI explains 86% of the variance in
mitochondrial 3-NT in untreated Tg, whereas Nr mice show no agerelated
pattern suggests that a regulatory mechanism acting in Nr
mice is dysregulated in Tg.
Homogenate versus mitochondrial 3-nitrotyrosine dichotomy
Complex and sometimes opposite relationships of 3-NT and
the MI also emerged for age-related patterns of homogenate
versus mitochondrial 3-NT. Because mitochondria are the
major source of superoxide, nitrative stress might be expected
to trace to mitochondria. Mitochondria indeed expressed
higher levels of 3-NT than homogenate in both Nr and Tg.
Regardless, we found remarkably opposite age-related patterns
of 3-NT in Tg mitochondria (linear decline) compared to
homogenate (linear increase) (Fig. 5). We previously found that
patterns of protein carbonyls (a marker of oxidative stress) also
showed different patterns in Tg homogenate (U-shaped) versus
mitochondria (a trend for age-related decline) (Aksenov et al.,
One possibility is that homogenate 3-NT mainly reflects
membrane-bound NAD(P)H oxidases and cytosolic nitric oxide
synthase. (Komatsu et al., 2006; Lemon et al., 2005; Rollo, 2007). A
further possibility is that mitochondrial turnover and/or declining
mitochondrial activity underlie the mitochondrial pattern, whereas
failure to remove damaged proteins (perhaps exacerbated by
ATP shortfalls) results in accumulating damage in the homogenate.
In the latter case, the association of the DSP with reduced
dichotomy in homogenate versus mitochondrial 3-NT (particularly
in Nr mice) could well reflect a benefit for the proteosome.
Differences in patterns of 3-NT compared to previous results for
protein carbonyls also suggest that various reactive oxygen species
may differ in their impacts with age. Regardless, maximal
homogenate carbonylation and nitosative stress converge in
senescent ages in Tg.
Levels of mitochondrial lipid peroxidation (4-HNE) were
positively associated with Mt 3-NT in normal mice (p < 0.003).
This was more pronounced in untreated Nr and ANCOVA (HNE
as covariate) detected a significant DSP*HNE interaction
(p < 0.01, Fig. 3). This could mean that the DSP partially acts
on Mt 3-NT by reducing levels of mitochondrial damage. Tg
showed a similar positive trend for Mt 3-NT with 4-HNE that
was not statistically resolved. Although the free radical and
mitochondrial theories of aging are under strong scrutiny,
these results suggest that more free radicals may be generated
by damaged mitochondria. In homogenate, Tg had 1.4-fold
higher levels of 4-HNE than Nr mice (p < 0.02) and rising levels
with age were similar in treated and untreated Nr mice and
resolved when Nr were pooled (p < 0.02).
Behavioral biomarkers of neuropathology
Tg are a model of accelerated cognitive aging (Aksenov et al.,
2010; Rollo et al., 1996) of likely relevance to Alzheimer’s,
Parkinson’s and Huntington’s (HD)
Collectively, our results provide a new model linking nitrative
stress to cognitive dysfunction and we demonstrate the utility of
Tg by exploring the potential of dietary supplements to modulate
reactive nitrogen processes in brain. The elevation of homogenate
3-NT in untreated Tg is profound whereas untreated Nr maintain
stable levels during aging. Our analyses demonstrate that various
aging biomarkers can express surprisingly complex non-linear
temporal patterns and spatial compartmentalization requiring
careful statistical dissection. Resolution requires samples spanning
the lifetime of groups. Tg are likely to prove valuable for
understanding neurodegenerative conditions associated with free
radical and nitrative stress (e.g., Alzheimer’s, Parkinson’s and
Huntington’s). Peroxynitrite is also implicated in many other
pathological processes including diabetes where amelioration
improves cardiac and vascular pathology (Obrosova et al., 2005).
Elevations in the GH axis are recognized as promoting aging
(Brown-Borg, 2009) and our results further confirm the transgenic
growth hormone mouse as a model of accelerated aging (Aksenov
et al., 2010; Lemon et al., 2003, 2005; Rollo et al., 1996).
This research was supported by a grant from the Natural
Sciences and Engineering Research Council of Canada to CDR, and
from the National Natural Science Foundation of China to JGL
(Grant No. 31070740). We also thank Dr. Brian McCarry (Chair of
Biology, McMaster University) for financial support for graduate
Disclosure statement: No actual or potential conflicts of
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