Age Related Changes in NAD+ Metabolism Oxidative Stress

Age Related Changes in NAD+ Metabolism Oxidative Stress

Introduction
Multiple degenerative processes are implicated in natural
senescence. As aging is associated with progressive decline in
organ function, elucidating the complex pathways controlling the
rate of aging is of significant clinical importance [1]. An important
mechanism contributing to aging is oxidative stress. The ‘‘freeradical
theory of aging’’, initially proposed by Harman (1956)
suggests that oxidative damage occurs with advanced aging due to
an imbalance between free radical and reactive species (ROS)
production, and cellular antioxidant defense mechanisms [2].
Elevated levels of intracellular ROS through hydrogen peroxide
treatment, or deficiency of ROS scavenging enzymes such as
superoxide dismutase (SOD1) knockdown, has been shown to
induce premature senescence and reduce cellular life span [3,4,5].
The mitochondria, represents the main producer of cellular
ROS in the human body, and approximately 1–2% of the oxygen
molecules consumed during normal respiration are converted into
highly reactive superoxide anion, which is rapidly dismutated to
H2O2 by the superoxide dismutases [6]. Other pathways and
events able to produce ROS include peroxisomal metabolism,
enzymatic synthesis of nitric oxide, phagocytic leukocytes, heat,
ultraviolet (UV) light, therapeutic drugs, and ionizing radiation
[7]. Intracellular ROS, due to their high reactivity, can interact
with a spectrum of biological molecules, leading to the oxidation of
several macromolecules, such as protein, lipids, and nucleic acids
[8]. As a result, vital functions, such as energy production,
maintenance of plasma membrane potential, and cellular ionic
homeostasis may be impaired in the early stage of oxidative stress
[8]. Excessive oxidative insult may also stimulate secondary events
leading to cell death via an apoptotic mechanism [9].
A major factor associated with age-related diseases is the increase of
oxidative DNA damage [7]. It is estimated that at least 5000 singlestranded
DNA breaks occur during a single cell cycle as a result of
ROS production [10,11]. Approximately 1%of these DNA breaks are
converted into double-stranded DNA breaks, primarily during DNA
replication. Accumulation of unrepaired DNA damage induced by
ROS can lead to arrest or induction of transcription, induction of signal
transduction pathways, replication errors and genomic instability
[10,11]. These molecular changes are observed in both cancer and
aging, and this supports the notion that chronic oxidative damage to
DNA might trigger cancer and promote aging [10,11].
The removal of oxidative DNA damage through repair of
DNA single strand breaks by DNA base excision repair,
PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19194
is facilitated by Poly(ADP-ribose) polymerase-1 (PARP)
[12,13,14]. PARP is an abundant protein modifying nuclear
enzyme involved in DNA repair. The enzymatic activity of
PARP is strongly activated in cells in response to treatment with
ROS such as H2O2 [15]. Activation of PARP leads to the
transfer of ADP-ribose moieties from NAD
+
, to the target
protein [16]. Since PARP uses NAD
+
as the only endogenous
substrate for poly-ADP-ribosylation, PARP activity is dependent
on the amount of NAD
+
available, and may act as a nuclear
energy sensor. Under physiological conditions, mild activation
of PARP can regulate several cellular processes, including DNA
repair, cell cycle progression, cell survival, chromatin remodeling,
and genomic stability [13,17]. However, overactivation of
PARP can repress genomic transcription and reduce cell
survival. NAD
+
, in addition to being a substrate for PARP,
also serves as an important redox carrier to power oxidative
phosphorylation and ATP production [18]. Depletion of NAD
+
following PARP hyperactivation has been shown to deplete
intracellular ATP stores leading to the release of apoptosisinducing
factors (AIF) and consequent cell death due to energy
restriction [19]. PARP activation has been implicated in the
pathogenesis of hypertension, atherosclerosis, lung injury,
haemorrhagic shock, and diabetic cardiovascular and kidney
complications [20,21,22,23,24]. In these diseases, the oxidantmediated
endothelial cell injury is dependent on PARP
activation, and can be attenuated by pharmacological PARP
inhibitors [25,26]. Therefore, tight regulation of PARP activity
is crucial to prevent the development of several age-related
pathological disorders.
In addition to its role in PARP activity, another essential
factor that is greatly affected by changes in intracellular NAD
+
levels is the class III histone deacetylases known as sirtuins, or
silent information regulator of gene transcription [27]. Gene
silencing by this family of enzymes has been correlated directly
with longer lifespan in yeast and worms [28]. In yeast, sir2 plays
a critical role in transcriptional silencing and maintenance of
genomic stability [29,30]. Sirt1 is the human homolog of sir2
and appears to be involved in several physiological functions
including the control of gene expression, cell cycle regulation,
apoptosis, DNA repair, metabolism, and aging [31,32,33]. Sirt1
can deacetylate numerous proteins such as tumor suppressor
protein, p53, which modulates various genes that control
damaged DNA [34]. The deacetylase activity of SIRT proteins
is dependent on the intracellular NAD
+
content [27]. They
catalyse a unique reaction that releases nicotinamide, acetyl
ADP-ribose (AADPR), and the deacetylated substrate [27].
Impaired SIRT1 activity due to PARP mediated NAD
+
depletion allows increased activity of several apoptotic effectors
such as p53, therefore sensitising cells to apoptosis. Adequate
NAD
+
levels are therefore critical to maintaining Sirt1 activity
which can delay apoptosis and provide vulnerable cells with
additional time to repair even after repeated exposure to
oxidative stress [35].
Though a number of studies have demonstrated elevated levels
of oxidative stress associated damage in aged tissue, to our
knowledge, no study has yet reported on changes in NAD
+
levels
during the aging process. In this study, we have characterized and
quantified changes in NAD
+
metabolism in the liver, heart,
kidney and lung from female wistar rats aged from 3 to 24
months, spanning life stages from young adulthood to old age
[36]. We quantified the levels of oxidative stress (in the form of
protein carbonyls, lipid peroxidation, and oxidative DNA
damage), total antioxidant and NAD
+
levels and PARP, Sirt1
and mitochondrial activities in these tissues at different stages of
life. Our results suggest that oxidative stress induced NAD
+
depletion could play a significant role in the aging process, by
compromising, energy production, DNA repair and genomic
surveillance.
Materials and Methods
Reagents and Chemicals
Phosphate buffer solution (PBS) was from Invitrogen (Melbourne,
Australia). Nicotinamide, bicine, b-nicotinamide
adenine dinucleotide reduced form (b-NADH), 3-[-4,5-dimethylthiazol-
2-yl]-2,5-diphenyl tetrazolium bromide (MTT),
alcohol dehydrogenase (ADH), sodium pyruvate, TRIS, cglobulins,
N-(1-naphthyl) ethylenediamine dihydrochloride,
EGTA, EDTA, tricarboxylic acid (TCA), Hepes, proteinase
K, Percoll, mannitol, 1,-dithio DL-threitol (DTT), KCN,
decylubiquinone, succinate, antimycin, rotenone, cytochrome
c, and sodium borohydride were obtained from Sigma-Aldrich
(Castle-Hill, Australia). Phenazine methosulfate (PMS) was
obtained from ICN Biochemicals (Ohio, USA). Bradford
reagent was obtained from BioRad (Hercules, CA, USA). DAPI
and polyclonal antibodies (pAb) for b-actin and all chemicals
used for Western blots (unless otherwise stated) were obtained
from Sigma-Aldrich (Castle-Hill, Australia). Polyclonal antibodies
for detection of (E)-4- Hydroxynonenal, Sirt1, phospho-
H2AX-ser139 were obtained from Alexis Biochemicals (San
Diego, CA, USA). Monoclonal antibody for the detection of
Poly(ADP-ribose) was purchased from Alexis Biochemicals (San
Diego, CA, USA). Polyclonal antibody for the detection of
acetylated p53 was obtained from Abcam (Cambridge, UK).
Monoclonal antibody to total p53 was obtained from Millipore
(Melbourne, Australia). Alexa 488- or Alexa 594-conjugated
anti-mouse IgG or anti-rabbit were purchased from Invitrogen.
All commercial antibodies were used at the concentrations
recommended by the manufacturer.

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